Spiro-lactam NMDA receptor modulators and uses thereof

ABSTRACT

Disclosed are compounds having enhanced potency in the modulation of NMDA receptor activity. Such compounds are contemplated for use in the treatment of conditions such as depression and related disorders. Orally available formulations and other pharmaceutically acceptable delivery forms of the compounds, including intravenous formulations, are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation patent application under 35 U.S.C. §120 of U.S. patent application Ser. No. 14/764,426, filed on Jul. 29,2015, which application is a U.S. national stage application under 35U.S.C. § 371 of International Application No. PCT/US2014/013639, filedon Jan. 29, 2014, which claims the benefit of and priority to U.S.Patent Application No. 61/757,942, filed on Jan. 29, 2013, each of whichis incorporated by reference herein in its entirety.

BACKGROUND

An N-methyl-d-aspartate (NMDA) receptor is a postsynaptic, ionotropicreceptor that is responsive to, inter cilia, the excitatory amino acidsglutamate and glycine and the synthetic compound NMDA. The NMDA receptorcontrols the flow of both divalent and monovalent ions into thepostsynaptic neural cell through a receptor associated channel (Fosteret al., Nature 1987, 329:395-396; Mayer et al., Trends in Pharmacol.Sci. 1990, 11:254-260). The NMDA receptor has been implicated duringdevelopment in specifying neuronal architecture and synapticconnectivity, and may be involved in experience-dependent synapticmodifications. In addition, NMDA receptors are also thought to beinvolved in long term potentiation and central nervous system disorders.

The NMDA receptor plays a major role in the synaptic plasticity thatunderlies many higher cognitive functions, such as memory acquisition,retention and learning, as well as in certain cognitive pathways and inthe perception of pain (Collingridge et al., The NMDA Receptor, OxfordUniversity Press, 1994). In addition, certain properties of NMDAreceptors suggest that they may be involved in theinformation-processing in the brain that underlies consciousness itself.

The NMDA receptor has drawn particular interest since it appears to beinvolved in a broad spectrum of CNS disorders. For instance, duringbrain ischemia caused by stroke or traumatic injury, excessive amountsof the excitatory amino acid glutamate are released from damaged oroxygen deprived neurons. This excess glutamate binds to the NMDAreceptors which opens their ligand-gated ion channels; in turn thecalcium influx produces a high level of intracellular calcium whichactivates a biochemical cascade resulting in protein degradation andcell death. This phenomenon, known as excitotoxicity, is also thought tobe responsible for the neurological damage associated with otherdisorders ranging from hypoglycemia and cardiac arrest to epilepsy. Inaddition, there are preliminary reports indicating similar involvementin the chronic neurodegeneration of Huntington's, Parkinson's, andAlzheimer's diseases. Activation of the NMDA receptor has been shown tobe responsible for post-stroke convulsions, and, in certain models ofepilepsy, activation of the NMDA receptor has been shown to be necessaryfor the generation of seizures. Neuropsychiatric involvement of the NMDAreceptor has also been recognized since blockage of the NMDA receptorCa⁺⁺ channel by the animal anesthetic PCP (phencyclidine) produces apsychotic state in humans similar to schizophrenia (reviewed in Johnson,K. and Jones, S., 1990). Further, NMDA receptors have also beenimplicated in certain types of spatial learning.

The NMDA receptor is believed to consist of several protein chainsembedded in the postsynaptic membrane. The first two types of subunitsdiscovered so far form a large extracellular region, which probablycontains most of the allosteric binding sites, several transmembraneregions looped and folded so as to form a pore or channel, which ispermeable to Ca⁺⁺, and a carboxyl terminal region. The opening andclosing of the channel is regulated by the binding of various ligands todomains (allosteric sites) of the protein residing on the extracellularsurface. The binding of the ligands is thought to affect aconformational change in the overall structure of the protein which isultimately reflected in the channel opening, partially opening,partially closing, or closing.

NMDA receptor compounds may exert dual (agonist/antagonist) effect onthe NMDA receptor through the allosteric sites. These compounds aretypically termed “partial agonists”. In the presence of the principalsite ligand, a partial agonist will displace some of the ligand and thusdecrease Ca⁺⁺ flow through the receptor. In the absence of or loweredlevel of the principal site ligand, the partial agonist acts to increaseCa⁺⁺ flow through the receptor channel.

A need continues to exist in the art for novel and more specific/potentcompounds that are capable of binding the glycine binding site of NMDAreceptors, and provide pharmaceutical benefits. In addition, a needcontinues to exist in the medical arts for orally deliverable forms ofsuch compounds.

SUMMARY

Provided herein, at least in part, are compounds that are NMDAmodulators, for example, partial agonists of NMDA. For example,disclosed herein are compounds represented by the formula:

and pharmaceutically acceptable salts, stereoisomers, and N-oxidesthereof, wherein

-   -   R_(b) is selected from the group consisting of H, halogen,        hydroxyl, cyano and C₁-C₆ alkyl;    -   R is H, C₁-C₆ alkyl or —C(O)—C₁-C₆ alkyl;    -   R₁ is H or C₁-C₆ alkyl;    -   R₂ is H or C₁-C₆ alkyl;    -   R₃ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆        cycloalkyl, phenyl, or a 4-6 membered heteroaryl with one, two        or three heteroatoms each selected from O, S or N, wherein R₃        may be optionally substituted with one two or three substituents        each selected from the group consisting of amino, halogen, C₁-C₆        alkyl, C₁-C₆ alkoxy, hydroxyl, phenyl (optionally substituted by        one, two or three substituents each independently selected from        R^(a)) or benzyl (optionally substituted by one two or three        substituents each independently selected from R^(a));    -   R^(a) is selected from the group consisting of halogen, C₁-C₆        alkyl (optionally substituted by one, two or three halogens),        C₃-C₆ cycloalkyl (optionally substituted by one, two or three        halogens), or C₁-C₆ alkoxy (optionally substituted by one, two        or three halogens);    -   R₄ is H or C₁-C₆ alkyl;    -   R₅ is H or C₁-C₆ alkyl;    -   X is selected from the group consisting of: H, C₁-C₆ alkyl, —OH,        C₁-C₆ alkoxy, —CO₂H, —C(O)NR^(c)R^(d), and a 4- to 6-membered        heteroaryl ring with one, two or three heteroatoms each selected        from O, S or N, wherein the heteroaryl ring may be optionally        substituted with one two or three substituents each selected        from the group consisting of halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy,        hydroxyl and phenyl; and    -   R^(c) and R^(d) are each independently selected from the group        consisting of H, C₁-C₆ alkyl, or phenyl, or together with the        nitrogen to which they are attached, form a 4-6 membered        heterocyclic ring, which may have an additional heteroatom        selected from O, S, or N; wherein the 4-6 membered heterocyclic        ring may optionally be substituted by one or more substituents        selected from the group consisting of halogen, cyano, oxo, and        C₁-C₆ alkyl;        or in other embodiments, the variables set forth in formula (I)        are defined as follows:

R_(b) is selected from the group consisting of H, halogen, hydroxyl,cyano and C₁-C₆alkyl (e.g., H);

R is H, C₁-C₆ alkyl or —C(O)—C₁-C₆alkyl;

R₁ is H or C₁-C₆ alkyl;

R₂ is H or C₁-C₆ alkyl;

R₃ is C₁-C₆ alkyl; C₁-C₆ alkoxy; —O—C₁-C₆ alkylene-phenyl; C₂-C₆alkenyl; C₂-C₆ alkynyl; C₃-C₆ cycloalkyl; phenyl; or heteroarylincluding from 5 to 6 ring atoms wherein 1, 2, or 3 of the ring atomsare independently selected from the group consisting of N, NH, N(C1-C3alkyl), O, and S, wherein R₃ is optionally substituted with one, two, orthree substituents independently selected from the group consisting ofamino, protected amino, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, hydroxyl,phenyl (optionally substituted by one, two, or three substituents eachindependently selected from R^(a)), benzyl (optionally substituted byone, two, or three substituents each independently selected from R^(a)),and —C₁-C₆ alkylene-C₃-C₆ cycloalkyl (optionally substituted by one, twoor three substituents independent selected from halogen and C₁-C₆alkyl);

R^(a) is selected from the group consisting of halogen, C₁-C₆alkyl(optionally substituted by one, two or three halogens), C₃-C₆ cycloalkyl(optionally substituted by one, two or three halogens), and C₁-C₆ alkoxy(optionally substituted by one, two or three halogens);

R₄ is H or C₁-C₆ alkyl;

R₅ is H or C₁-C₆ alkyl;

X is selected from the group consisting of: H; C₁-C₆ alkyl; —OH; C₁-C₆alkoxy; —CO₂H; —C(O)NR^(c)R^(d); and heteroaryl including from 5 to 6ring atoms wherein 1, 2, or 3 of the ring atoms are independentlyselected from the group consisting of N, NH, N(C1-C3 alkyl), O, and S,wherein the heteroaryl ring may be optionally substituted with one, two,or three substituents independently selected from the group consistingof halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, hydroxyl and phenyl; and

R^(c) and R^(d) are each independently selected from the groupconsisting of H, C₁-C₆ alkyl, or phenyl, or R^(c) and R^(d) togetherwith the nitrogen to which they are attached, form heterocyclylincluding from 4 to 6 ring atoms; wherein the heterocyclyl includes notmore than two ring heteroatoms (including the nitrogen atom attached toR^(c) and R^(d)), and the second ring heteroatom, when present, isindependently selected from the group consisting of N, NH, N(C1-C3alkyl), O, and S; and wherein the heterocyclyl is optionally substitutedwith from 1-3 substituents independently selected from the groupconsisting of halogen, cyano, oxo, and C₁-C₆ alkyl.

Also provided herein, at least in part, are compounds that are NMDAmodulators, for example, partial agonists of NMDA. For example,disclosed herein are compounds represented by the formula:

and pharmaceutically acceptable salts, stereoisomers, and N-oxidesthereof, wherein

R_(b) is selected from the group consisting of H, halogen, hydroxyl,cyano and C₁-C₆alkyl;

R is H, C₁-C₆ alkyl or —C(O)—C₁-C₆alkyl;

R₁ is H or C₁-C₆ alkyl;

R₂ is H or C₁-C₆ alkyl;

R₄ is H or C₁-C₆ alkyl;

R₅ is H or C₁-C₆ alkyl;

X is selected from the group consisting of: H; C₁-C₆ alkyl; —OH; C₁-C₆alkoxy; —CO₂H; —C(O)NR^(c)R^(d); and heteroaryl including from 5 to 6ring atoms wherein 1, 2, or 3 of the ring atoms are independentlyselected from the group consisting of N, NH, N(C1-C3 alkyl), O, and S,wherein the heteroaryl ring may be optionally substituted with one, two,or three substituents independently selected from the group consistingof halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, hydroxyl and phenyl; and

R^(c) and R^(d) are each independently selected from the groupconsisting of H, C₁-C₆ alkyl, or phenyl, or R^(c) and R^(d) togetherwith the nitrogen to which they are attached, form heterocyclylincluding from 4 to 6 ring atoms; wherein the heterocyclyl includes notmore than two ring heteroatoms (including the nitrogen atom attached toR^(c) and R^(d)), and the second ring heteroatom, when present, isindependently selected from the group consisting of N, NH, N(C1-C3alkyl), O, and S; and wherein the heterocyclyl is optionally substitutedwith from 1-3 substituents independently selected from the groupconsisting of halogen, cyano, oxo, and C₁-C₆ alkyl.

Also provided herein are pharmaceutically acceptable compositionscomprising a disclosed compound, and a pharmaceutically acceptableexcipient. For example, such compositions may be suitable for oral orintravenous administration to a patient.

In another aspect, a method of treating a condition selected from thegroup consisting of autism, anxiety, depression, bipolar disorder,attention deficit disorder, attention deficit hyperactivity disorder(ADHD), schizophrenia, a psychotic disorder, a psychotic symptom, socialwithdrawal, obsessive-compulsive disorder, phobia, post-traumatic stresssyndrome, a behavior disorder, an impulse control disorder, a substanceabuse disorder, a sleep disorder, a memory disorder, a learningdisorder, urinary incontinence, multiple system atrophy, progressivesupra-nuclear palsy, Friedrich's ataxia, Down's syndrome, fragile Xsyndrome, tuberous sclerosis, olivio-ponto-cerebellar atrophy, cerebralpalsy, drug-induced optic neuritis, ischemic retinopathy, diabeticretinopathy, glaucoma, dementia, AIDS dementia, Alzheimer's disease,Huntington's chorea, spasticity, myoclonus, muscle spasm, Tourette'ssyndrome, epilepsy, cerebral ischemia, stroke, a brain tumor, traumaticbrain injury, cardiac arrest, myelopathy, spinal cord injury, peripheralneuropathy, acute neuropathic pain, and chronic neuropathic, in apatient in need thereof is provided. Such methods may compriseadministering to the patient a pharmaceutically effective amount of adisclosed compound or pharmaceutically acceptable salts, stereoisomers,N-oxides, and hydrates thereof.

In some embodiments, a contemplated method includes treating depression.For example, depression may include one or more of major depressivedisorder, dysthymic disorder, psychotic depression, postpartumdepression, seasonal affective disorder, bipolar disorder, mooddisorder, or depression caused by a chronic medical condition. In otherembodiments, a contemplated method may treat schizophrenia. Suchschizophrenia may be, for example, paranoid type schizophrenia,disorganized type schizophrenia, catatonic type schizophrenia,undifferentiated type schizophrenia, residual type schizophrenia,post-schizophrenic depression, or simple schizophrenia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the potentiation of [³H]MK-801 binding in the presence ofCompound B.

FIG. 2 shows the potentiation of [³H]MK-801 binding in the presence ofCompound C.

FIG. 3 shows the potentiation of [³H]MK-801 binding in the presence ofCompound D.

FIG. 4 shows the potentiation of [³H]MK-801 binding in the presence ofCompound H.

FIG. 5 shows results of long term potentiation in hippocampal slicesusing Compound B.

DETAILED DESCRIPTION

This disclosure is generally directed to compounds that are capable ofmodulating NMDA, e.g., NMDA antagonists or partial agonists, andcompositions and/or methods of using the disclosed compounds.

Definitions

“Treating” includes any effect, e.g., lessening, reducing, modulating,or eliminating, that results in the improvement of the condition,disease, disorder and the like.

The term “alkenyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon double bond, suchas a straight or branched group of 2-6 or 3-4 carbon atoms, referred toherein for example as C₂-C₆ alkenyl, and C₃-C₄ alkenyl, respectively.Exemplary alkenyl groups include, but are not limited to, vinyl, allyl,butenyl, pentenyl, etc.

The term “alkoxy” as used herein refers to a straight or branched alkylgroup attached to an oxygen (alkyl-O—). Exemplary alkoxy groups include,but are not limited to, alkoxys of 1-6 or 2-6 carbon atoms, referred toherein as C₁-C₆ alkoxy, and C₂-C₆ alkoxy, respectively. Exemplary alkoxygroups include, but are not limited to methoxy, ethoxy, isopropoxy, etc.

The term “alkenyloxy” used herein refers to a straight or branchedalkenyl group attached to an oxygen (alkenyl-O). Exemplary alkenoxygroup include, but are not limited to, groups with an alkenyl group of3-6 carbon atoms, (also e.g. referred to as C₃-C₆ alkenyloxy). Exemplary“alkenoxy” groups include, but are not limited to allyloxy, butenyloxy,etc.

The term “alkynyloxy” used herein refers to a straight or branchedalkynyl group attached to an oxygen (alkynyl-O)). Exemplary alkynyloxygroups include, but are not limited to, C₃-C₆alkynyloxy, e.g.,propynyloxy.

The term “alkyl” as used herein refers to a saturated straight orbranched hydrocarbon, such as a straight or branched group of 1-6, 1-4,or 1-3 carbon atoms, referred to herein as C₁-C₆ alkyl, C₁-C₄ alkyl, andC₁-C₃ alkyl, respectively. Exemplary alkyl groups include, but are notlimited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl,2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 3-methyl-2-butyl,2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl,4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl,2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl,hexyl, etc. The term “haloalkyl” as used herein refers to a saturatedstraight or branched alkyl groups, in which one or more hydrogen atomsof the alkyl group are replaced with one or more independently selectedhalogens. The term “haloalkyl” encompasses alkyl groups in which all ofhydrogen atoms of the alkyl group are replaced independently selectedhalogens (sometimes referred to as “perhalo” alkyl groups. Exemplaryhaloalkyl groups include, but are not limited to, CH₂F, CH₂CH₂Cl, CF₃,CHFCH₂Cl.

The term “alkynyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon triple bond, suchas a straight or branched group of 2-6, or 3-6 carbon atoms, referred toherein as C₂-C₆ alkynyl, and C₃-C₆ alkynyl, respectively. Exemplaryalkynyl groups include, but are not limited to, ethynyl, propynyl,butynyl, pentynyl, hexynyl, methylpropynyl, etc.

The term “bridged cycloalkyl”, as used herein, is defined as amonocyclic 4- to 7-membered cycloalkyl group in which two non-adjacentatoms are linked by a CH₂ or CH₂CH₂ group. A “bridged cycloalkyl” may befused to one or more phenyl, partially unsaturated, or saturated rings.Examples of bridged carbocyclic groups include but are not limited tobicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[2.2.2]octene etc.

The term “carbonyl” as used herein refers to the radical —C(O)—. Theterm “cyano” as used herein refers to the radical —CN. The term “nitro”refers to the radical —NO₂. The term “H” refers to hydrogen.

The term “cycloalkoxy” as used herein refers to a cycloalkyl groupattached to an oxygen (cycloalkyl-O—).

The term “cycloalkyl” as used herein refers to a monocyclic saturated orpartially unsaturated hydrocarbon group of for example 3-6, or 4-6carbons, referred to herein, e.g., as “C₃₋₆cycloalkyl” or“C₄₋₆cycloalkyl,” and derived from a cycloalkane. Exemplary cycloalkylgroups include, but are not limited to, cyclohexyl, cyclohexenyl,cyclopentyl, cyclobutyl, cyclopropyl or cyclopentyl.

The terms “halo” or “halogen” as used herein refer to F, Cl, Br, or I.

The terms “heteroaryl” as used herein refers to a monocyclic aromatic4-6 membered ring system containing one or more heteroatoms, for exampleone to three heteroatoms, such as nitrogen, oxygen, and sulfur. Wherepossible, said heteroaryl ring may be linked to the adjacent radicalthough carbon or nitrogen. Examples of heteroaryl rings include but arenot limited to furyl, thienyl, pyrrolyl, thiazolyl, oxazolyl,isothiazolyl, isoxazolyl, imidazolyl, pyrazolyl, triazolyl, oxadiazolyl(e.g., 1,2,4-oxadiazolyl or 1,3,4-oxadiazolyl), pyridyl, andpyrimidinyl.

The terms “heterocyclyl” or “heterocyclic group” are art-recognized andrefer to saturated or partially unsaturated 4- to 7-membered ringstructures, whose ring structures include one to three heteroatoms, suchas nitrogen, oxygen, and sulfur. A heterocycle may be fused to one ormore phenyl, partially unsaturated, or saturated rings. Examples ofheterocyclyl groups include but are not limited to pyrrolidine,piperidine, morpholine, thiomorpholine, and piperazine.

The term “heterocyclylalkoxy” as used herein refers to aheterocyclyl-alkyl-O— group.

The term “heterocyclyloxyalkyl” refers to a heterocyclyl-O-alkyl- group.

The term “heterocycloxy” refers to a heterocyclyl-O— group. The term“cycloalkyloxy” refers to a cycloalkyl-O— group.

The term “heteroaryloxy” refers to a heteroaryl-O— group.

The terms “hydroxy” and “hydroxyl” as used herein refers to the radical—OH.

The term “oxo” as used herein refers to the radical ═O.

The terms “nitrogen protecting group” or “amino protecting group” isart-recognized and as used herein refers to a chemical moiety that iscovalently linked to a nitrogen atom of an amino (primary or secondary)group and that temporarily blocks (protects) the reactivity of the aminogroup during a synthetic step and is selectively removed once thesynthetic step is complete. Nitrogen protecting groups include, forexample, 9-Fluorenylmethyloxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc),carbobenzyloxycarbonyl (Cbz), p-methoxybenzyloxycarbonyl, acetyl,trifluoroacetyl, benzoyl, phthalimido, benzyl (Bn), p-methoxybenzyl,p-methoxyphenyl, 3,4-dimethoxybenzyl, triphenylmethyl, benzylidene, andp-toluenesulfonyl (Ts). In some embodiments, the nitrogen protectinggroup can have one of the following formulas: C(O)OR₃₁ or C(O)R₃₂ asdefined herein. In certain embodiments, R₃₁ is selected from the groupconsisting of: C₁-C₆ alkyl; C₁-C₆ haloalkyl; C₂-C₆ alkenyl; C₂-C₆alkynyl; C₃-C₁₀ cycloalkyl, wherein the C₃-C₁₀ cycloalkyl is optionallysubstituted with from 1-3 independently selected C₁-C₃ alkyl;—CH₂—C₃-C₁₀ cycloalkyl wherein the C₃-C₁₀ cycloalkyl is optionallysubstituted with from 1-3 independently selected C₁-C₃ alkyl; —CH₂—phenyl, wherein the phenyl is optionally substituted with from 1-2substituents independently selected from C₁-C₃ alkyl, C₁-C₃ haloalkyl,C₁-C₃ alkoxy, C₁-C₃ haloalkoxy, nitro, halo, SO₂Me, cyano, and—OC(O)CH₃; and —CH₂-pyridyl. In certain embodiments, R₃₂ is selectedfrom the group consisting of: H; C₁-C₆ alkyl; C₁-C₆ haloalkyl; phenyl,wherein the phenyl is optionally substituted with from 1-2 substituentsindependently selected from C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃ alkoxy,C₁-C₃ haloalkoxy, nitro, halo, SO₂Me, cyano, and —OC(O)CH₃; and pyridyl.

As used in the present disclosure, the term “partial NMDA receptoragonist” generally refers to a compound that is capable of binding to aglycine binding site of an NMDA receptor; at low concentrations a NMDAreceptor agonist acts substantially as agonist and at highconcentrations it acts substantially as an antagonist. Theseconcentrations are experimentally determined for each and every “partialagonist.

“Pharmaceutically or pharmacologically acceptable” include molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate. For human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” as used herein refers to any and all solvents,dispersion media, coatings, isotonic and absorption delaying agents, andthe like, that are compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. The compositions may also contain other activecompounds providing supplemental, additional, or enhanced therapeuticfunctions.

The term “pharmaceutical composition” as used herein refers to acomposition comprising at least one compound as disclosed hereinformulated together with one or more pharmaceutically acceptablecarriers.

“Individual,” “patient,” or “subject” are used interchangeably andinclude any animal, including mammals, preferably mice, rats, otherrodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates,and most preferably humans. The compounds of the invention can beadministered to a mammal, such as a human, but can also be administeredto other mammals such as an animal in need of veterinary treatment,e.g., domestic animals (e.g., dogs, cats, and the like), farm animals(e.g., cows, sheep, pigs, horses, and the like) and laboratory animals(e.g., rats, mice, guinea pigs, and the like). The mammal treated in themethods of the invention is desirably a mammal in which treatment e.g.,of pain or depression is desired. “Modulation” includes antagonism(e.g., inhibition), agonism, partial antagonism and/or partial agonism.

In the present specification, the term “therapeutically effectiveamount” means the amount of the subject compound that will elicit thebiological or medical response of a tissue, system, animal or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician. The compounds of the invention are administered intherapeutically effective amounts to treat a disease. Alternatively, atherapeutically effective amount of a compound is the quantity requiredto achieve a desired therapeutic and/or prophylactic effect, such as anamount which results in lessening a symptom of depression.

The term “pharmaceutically acceptable salt(s)” as used herein refers tosalts of acidic or basic groups that may be present in compounds used inthe present compositions. Compounds included in the present compositionsthat are basic in nature are capable of forming a wide variety of saltswith various inorganic and organic acids. The acids that may be used toprepare pharmaceutically acceptable acid addition salts of such basiccompounds are those that form non-toxic acid addition salts, i.e., saltscontaining pharmacologically acceptable anions, including but notlimited to malate, oxalate, chloride, bromide, iodide, nitrate, sulfate,bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate,salicylate, citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.Compounds included in the present compositions that are acidic in natureare capable of forming base salts with various pharmacologicallyacceptable cations. Examples of such salts include alkali metal oralkaline earth metal salts and, particularly, calcium, magnesium,sodium, lithium, zinc, potassium, and iron salts. Compounds included inthe present compositions that include a basic or acidic moiety may alsoform pharmaceutically acceptable salts with various amino acids. Thecompounds of the disclosure may contain both acidic and basic groups;for example, one amino and one carboxylic acid group. In such a case,the compound can exist as an acid addition salt, a zwitterion, or a basesalt.

The compounds of the disclosure may contain one or more chiral centersand/or double bonds and, therefore, exist as stereoisomers, such asgeometric isomers, enantiomers or diastereomers. The term“stereoisomers” when used herein consist of all geometric isomers,enantiomers or diastereomers. These compounds may be designated by thesymbols “R” or “S,” depending on the configuration of substituentsaround the stereogenic carbon atom. The present invention encompassesvarious stereoisomers of these compounds and mixtures thereof.Stereoisomers include enantiomers and diastereomers. Mixtures ofenantiomers or diastereomers may be designated “(±)” in nomenclature,but the skilled artisan will recognize that a structure may denote achiral center implicitly.

The compounds of the disclosure may contain one or more chiral centersand/or double bonds and, therefore, exist as geometric isomers,enantiomers or diastereomers. The enantiomer and diastereomers may bedesignated by the symbols “(+),” “(−).” “R” or “S,” depending on theconfiguration of substituents around the stereogenic carbon atom, butthe skilled artisan will recognize that a structure may denote a chiralcenter implicitly. Geometric isomers, resulting from the arrangement ofsubstituents around a carbon-carbon double bond or arrangement ofsubstituents around a cycloalkyl or heterocyclic ring, can also exist inthe compounds of the present invention. The symbol

denotes a bond that may be a single, double or triple bond as describedherein. Substituents around a carbon-carbon double bond are designatedas being in the “Z” or “E” configuration wherein the terms “Z” and “E”are used in accordance with IUPAC standards. Unless otherwise specified,structures depicting double bonds encompass both the “E” and “Z”isomers. Substituents around a carbon-carbon double bond alternativelycan be referred to as “cis” or “trans,” where “cis” representssubstituents on the same side of the double bond and “trans” representssubstituents on opposite sides of the double bond. The arrangement ofsubstituents around a carbocyclic ring can also be designated as “cis”or “trans.” The term “cis” represents substituents on the same side ofthe plane of the ring and the term “trans” represents substituents onopposite sides of the plane of the ring. Mixtures of compounds whereinthe substituents are disposed on both the same and opposite sides ofplane of the ring are designated “cis/trans.”

The term “stereoisomers” when used herein consist of all geometricisomers, enantiomers or diastereomers. The present invention encompassesvarious stereoisomers of these compounds and mixtures thereof.

Individual enantiomers and diasteriomers of compounds of the presentinvention can be prepared synthetically from commercially availablestarting materials that contain asymmetric or stereogenic centers, or bypreparation of racemic mixtures followed by resolution methods wellknown to those of ordinary skill in the art. These methods of resolutionare exemplified by (1) attachment of a mixture of enantiomers to achiral auxiliary, separation of the resulting mixture of diastereomersby recrystallization or chromatography and liberation of the opticallypure product from the auxiliary, (2) salt formation employing anoptically active resolving agent, (3) direct separation of the mixtureof optical enantiomers on chiral liquid chromatographic columns or (4)kinetic resolution using stereoselective chemical or enzymatic reagents.Racemic mixtures can also be resolved into their component enantiomersby well-known methods, such as chiral-phase gas chromatography orcrystallizing the compound in a chiral solvent. Stereoselectivesyntheses, a chemical or enzymatic reaction in which a single reactantforms an unequal mixture of stereoisomers during the creation of a newstereocenter or during the transformation of a pre-existing one, arewell known in the art. Stereoselective syntheses encompass both enantio-and diastereoselective transformations. For examples, see Carreira andKvaerno, Classics in Stereoselective Synthesis, Wiley-VCH: Weinheim,2009.

The compounds disclosed herein can exist in solvated as well asunsolvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms. In one embodiment, thecompound is amorphous. In one embodiment, the compound is a singlepolymorph. In another embodiment, the compound is a mixture ofpolymorphs. In another embodiment, the compound is in a crystallineform.

The invention also embraces isotopically labeled compounds of theinvention which are identical to those recited herein, except that oneor more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorus, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O,¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively. For example, a compoundof the invention may have one or more H atom replaced with deuterium.

Certain isotopically-labeled disclosed compounds (e.g., those labeledwith ³H and ¹⁴C) are useful in compound and/or substrate tissuedistribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C)isotopes are particularly preferred for their ease of preparation anddetectability. Further, substitution with heavier isotopes such asdeuterium (i.e., ²H) may afford certain therapeutic advantages resultingfrom greater metabolic stability (e.g., increased in vivo half-life orreduced dosage requirements) and hence may be preferred in somecircumstances. Isotopically labeled compounds of the invention cangenerally be prepared by following procedures analogous to thosedisclosed in the e.g., Examples herein by substituting an isotopicallylabeled reagent for a non-isotopically labeled reagent.

The term “prodrug” refers to compounds that are transformed in vivo toyield a disclosed compound or a pharmaceutically acceptable salt,hydrate or solvate of the compound. The transformation may occur byvarious mechanisms (such as by esterase, amidase, phosphatase, oxidativeand or reductive metabolism) in various locations (such as in theintestinal lumen or upon transit of the intestine, blood or liver).Prodrugs are well known in the art (for example, see Rautio,Kumpulainen, et al, Nature Reviews Drug Discovery 2008, 7, 255). Forexample, if a compound of the invention or a pharmaceutically acceptablesalt, hydrate or solvate of the compound contains a carboxylic acidfunctional group, a prodrug can comprise an ester formed by thereplacement of the hydrogen atom of the acid group with a group such as(C₁-C₈)alkyl, (C₂-C₁₂)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl havingfrom 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having from 5to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbonatoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms,1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms,N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms,1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms,3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl,di-N,N—(C₁-C₂)alkylamino(C₂-C₃)alkyl (such as β-dimethylaminoethyl),carbamoyl-(C₁-C₂)alkyl, N,N-di(C₁-C₂)alkylcarbamoyl-(C₁-C₂)alkyl andpiperidino-, pyrrolidino- or morpholino(C₂-C₃)alkyl.

Similarly, if a compound of the invention contains an alcohol functionalgroup, a prodrug can be formed by the replacement of the hydrogen atomof the alcohol group with a group such as (C₁-C₆)alkanoyloxymethyl,1-((C₁-C₆)alkanoyloxy)ethyl, 1-methyl-1-((C₁-C₆)alkanoyloxy)ethyl(C₁-C₆)alkoxycarbonyloxymethyl, N—(C₁-C₆)alkoxycarbonylaminomethyl,succinoyl, (C₁-C₆)alkanoyl, α-amino(C₁-C₄)alkanoyl, arylacyl andα-aminoacyl, or α-aminoacyl-α-aminoacyl, where each α-aminoacyl group isindependently selected from the naturally occurring L-amino acids,P(O)(OH)₂, —P(O)(O(C₁-C₆)alkyl)₂ or glycosyl (the radical resulting fromthe removal of a hydroxyl group of the hemiacetal form of acarbohydrate).

If a compound of the invention incorporates an amine functional group, aprodrug can be formed, for example, by creation of an amide orcarbamate, an N-acyloxyakyl derivative, an (oxodioxolenyl)methylderivative, an N-Mannich base, imine or enamine. In addition, asecondary amine can be metabolically cleaved to generate a bioactiveprimary amine, or a tertiary amine can metabolically cleaved to generatea bioactive primary or secondary amine. For examples, see Simplicio, etal., Molecules 2008, 13, 519 and references therein.

Compounds

Disclosed compounds include those represented by the formula:

and pharmaceutically acceptable salts, stereoisomers, and N-oxidesthereof, wherein

-   -   R_(b) is selected from the group consisting of H, halogen,        hydroxyl, cyano and C₁-C₆ alkyl;    -   R is H, C₁-C₆ alkyl or —C(O)—C₁-C₆ alkyl;    -   R₁ is H or C₁-C₆ alkyl;    -   R₂ is H or C₁-C₆ alkyl;    -   R₃ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆        cycloalkyl, phenyl, or a 4-6 membered heteroaryl with one, two        or three heteroatoms each selected from O, S or N, wherein R₃        may be optionally substituted with one two or three substituents        each selected from the group consisting of amino, halogen, C₁-C₆        alkyl, C₁-C₆ alkoxy, hydroxyl, phenyl (optionally substituted by        one, two or three substituents each independently selected from        R^(a)) or benzyl (optionally substituted by one two or three        substituents each independently selected from R^(a));    -   R^(a) is selected from the group consisting of halogen,        C₁-C₆alkyl (optionally substituted by one, two or three        halogens), C₃-C₆ cycloalkyl (optionally substituted by one, two        or three halogens), or C₁-C₆ alkoxy (optionally substituted by        one, two or three halogens);    -   R₄ is H or C₁-C₆ alkyl;    -   R₅ is H or C₁-C₆ alkyl;    -   X is selected from the group consisting of: H, C₁-C₆ alkyl, —OH,        C₁-C₆ alkoxy, —CO₂H, —C(O)NR^(c)R^(d), and a 4- to 6-membered        heteroaryl ring with one, two or three heteroatoms each selected        from O, S or N, wherein the heteroaryl ring may be optionally        substituted with one two or three substituents each selected        from the group consisting of halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy,        hydroxyl and phenyl; and    -   R^(c) and R^(d) are each independently selected from the group        consisting of H, C₁-C₆ alkyl, or phenyl, or together with the        nitrogen to which they are attached, form a 4-6 membered        heterocyclic ring, which may have an additional heteroatom        selected from O, S, or N; wherein the 4-6 membered heterocyclic        ring may optionally be substituted by one or more substituents        selected from the group consisting of halogen, cyano, oxo, and        C₁-C₆ alkyl;        or in other embodiments, the variables set forth in formula (I)        are defined as follows:

R_(b) is selected from the group consisting of H, halogen, hydroxyl,cyano and C₁-C₆alkyl (e.g., H);

R is H, C₁-C₆ alkyl or —C(O)—C₁-C₆alkyl;

R₁ is H or C₁-C₆ alkyl;

R₂ is H or C₁-C₆ alkyl;

R₃ is C₁-C₆ alkyl; C₁-C₆ alkoxy; —O—C₁-C₆ alkylene-phenyl; C₂-C₆alkenyl; C₂-C₆ alkynyl; C₃-C₆ cycloalkyl; phenyl; or heteroarylincluding from 5 to 6 ring atoms wherein 1, 2, or 3 of the ring atomsare independently selected from the group consisting of N, NH, N(C₁-C₃alkyl), O, and S, wherein R₃ is optionally substituted with one, two, orthree substituents independently selected from the group consisting ofamino, protected amino, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, hydroxyl,phenyl (optionally substituted by one, two, or three substituents eachindependently selected from R^(a)), benzyl (optionally substituted byone, two, or three substituents each independently selected from R^(a)),and —C₁-C₆ alkylene-C₃-C₆cycloalkyl (optionally substituted by one, twoor three substituents independent selected from halogen and C₁-C₆alkyl);

R^(a) is selected from the group consisting of halogen, C₁-C₆alkyl(optionally substituted by one, two or three halogens), C₃-C₆ cycloalkyl(optionally substituted by one, two or three halogens), and C₁-C₆ alkoxy(optionally substituted by one, two or three halogens);

R₄ is H or C₁-C₆ alkyl;

R₅ is H or C₁-C₆ alkyl;

X is selected from the group consisting of: H; C₁-C₆ alkyl; —OH; C₁-C₆alkoxy; —CO₂H; —C(O)NR^(c)R^(d); and heteroaryl including from 5 to 6ring atoms wherein 1, 2, or 3 of the ring atoms are independentlyselected from the group consisting of N, NH, N(C1-C3 alkyl), O, and S,wherein the heteroaryl ring may be optionally substituted with one, two,or three substituents independently selected from the group consistingof halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, hydroxyl and phenyl; and

R^(c) and R^(d) are each independently selected from the groupconsisting of H, C₁-C₆ alkyl, or phenyl, or R^(c) and R^(d) togetherwith the nitrogen to which they are attached, form heterocyclylincluding from 4 to 6 ring atoms; wherein the heterocyclyl includes notmore than two ring heteroatoms (including the nitrogen atom attached toR^(c) and R^(d)), and the second ring heteroatom, when present, isindependently selected from the group consisting of N, NH, N(C1-C3alkyl), O, and S; and wherein the heterocyclyl is optionally substitutedwith from 1-3 substituents independently selected from the groupconsisting of halogen, cyano, oxo, and C₁-C₆ alkyl.

Also provided herein, at least in part, are compounds that are NMDAmodulators, for example, partial agonists of NMDA. For example,disclosed herein are compounds represented by the formula:

and pharmaceutically acceptable salts, stereoisomers, and N-oxidesthereof, wherein

R_(b) is selected from the group consisting of H, halogen, hydroxyl,cyano and C₁-C₆alkyl;

R is H, C₁-C₆ alkyl or —C(O)—C₁-C₆alkyl;

R₁ is H or C₁-C₆ alkyl;

R₂ is H or C₁-C₆ alkyl;

R₄ is H or C₁-C₆ alkyl;

R₅ is H or C₁-C₆ alkyl;

X is selected from the group consisting of: H; C₁-C₆ alkyl; —OH; C₁-C₆alkoxy; —CO₂H; —C(O)NR^(c)R^(d); and heteroaryl including from 5 to 6ring atoms wherein 1, 2, or 3 of the ring atoms are independentlyselected from the group consisting of N, NH, N(C₁-C₃ alkyl), O, and S,wherein the heteroaryl ring may be optionally substituted with one, two,or three substituents independently selected from the group consistingof halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, hydroxyl and phenyl; and

R^(c) and R^(d) are each independently selected from the groupconsisting of H, C₁-C₆ alkyl, or phenyl, or R^(c) and R^(d) togetherwith the nitrogen to which they are attached, form heterocyclylincluding from 4 to 6 ring atoms; wherein the heterocyclyl includes notmore than two ring heteroatoms (including the nitrogen atom attached toR^(c) and R^(d)), and the second ring heteroatom, when present, isindependently selected from the group consisting of N, NH, N(C1-C3alkyl), O, and S; and wherein the heterocyclyl is optionally substitutedwith from 1-3 substituents independently selected from the groupconsisting of halogen, cyano, oxo, and C₁-C₆ alkyl.

In some embodiments, R₁ is H.

In some embodiments, R₂ is H.

In some embodiments, R₃ is unsubstituted C₁-C₆ alkyl. In certainembodiments, R₃ is methyl.

In some embodiments, R₃ is C₁-C₆ alkyl substituted with one, two orthree substituents each selected from the group consisting of amino,halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, hydroxyl, phenyl (optionallysubstituted by one two or three substituents each independently selectedfrom R^(a)) and benzyl (optionally substituted by one two or threesubstituents each independently selected from R^(a)). In someembodiments, R₃ is C₁-C₆ alkyl substituted with one, two or threesubstituents each selected from the group consisting of amino, protectedamino, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, hydroxyl, phenyl (optionallysubstituted by one, two, or three substituents each independentlyselected from R^(a)), benzyl (optionally substituted by one two or threesubstituents each independently selected from R^(a)), and —C₁-C₆alkylene-C₃-C₆ cycloalkyl (optionally substituted by one, two or threesubstituents independently selected from halogen and C₁-C₆ alkyl). Incertain embodiments, R₃ is

In certain embodiments, R₃ is heteroaryl. In some embodiments, R₃ isheteroaryl including from 5 to 6 ring atoms wherein 1, 2, or 3 of thering atoms are independently selected from the group consisting of N,NH, N(C1-C3 alkyl), O, and S; which is optionally substituted with one,two, or three substituents independently selected from the groupconsisting of amino, protected amino, halogen, C₁-C₆ alkyl, C₁-C₆alkoxy, hydroxyl, phenyl (optionally substituted by one, two or threesubstituents each independently selected from R^(a)), benzyl (optionallysubstituted by one two or three substituents each independently selectedfrom R^(a)), and —C₁-C₆ alkylene-C₃-C₆cycloalkyl (optionally substitutedby one, two or three substituents independent selected from halogen andC₁-C₆ alkyl). In certain embodiments, R₃ (here, heteroaryl) isoptionally substituted with one, two, or three substituentsindependently selected from the group consisting of C₁-C₆ alkyl (e.g.,CH₃) and benzyl (optionally substituted by one two or three substituentseach independently selected from R^(a)). In certain embodiments, R₃(here, heteroaryl) is optionally substituted with one, two, or threesubstituents independently selected from the group consisting of CH₃ andbenzyl. In some embodiments, R₃ is selected from the group consisting of—CH₃,

In some embodiments, R₃ is —O—C₁-C₆ alkylene-phenyl (e.g.,—O—CH₂-phenyl), which is optionally substituted with one, two, or threesubstituents independently selected from the group consisting of amino,protected amino, halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, hydroxyl, phenyl(optionally substituted by one, two or three substituents eachindependently selected from R^(a)), benzyl (optionally substituted byone two or three substituents each independently selected from R^(a)),and —C₁-C₆ alkylene-C₃-C₆ cycloalkyl (optionally substituted by one, twoor three substituents independent selected from halogen and C₁-C₆alkyl). In certain embodiments, R₃ is benzyloxy.

In some embodiments, X is —C(O)NR^(c)R^(d). In certain embodiments,R^(c) and R^(d) are each independently selected from the groupconsisting of H, C₁-C₆ alkyl, and phenyl. For example, R^(c) and R^(d)are both H.

In other embodiments, X is a 4- to 6-membered heteroaryl ring. In someembodiments, X is heteroaryl including from 5 to 6 ring atoms wherein 1,2, or 3 of the ring atoms are independently selected from the groupconsisting of N, NH, N(C1-C3 alkyl), O, and S, which is optionallysubstituted with one, two, or three substituents independently selectedfrom the group consisting of halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy,hydroxyl and phenyl. In certain embodiments, X is selected from thegroup consisting of 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,4-triazolyl (optionally substituted with from 1-2 independentlyselected C₁-C₆ alkyl), pyridyl, and pyrimidinyl.

In some embodiments (including any of the foregoing embodimentsdescribed above), R is H or —C(O)—C₁-C₆alkyl (e.g., —C(O)—CH₃). In someembodiments (including any of the foregoing embodiments describedabove), R₅ is C₁-C₆ alkyl (e.g., —CH₃). In some embodiments (includingany of the foregoing embodiments described above), R₄ is H. In someembodiments (including any of the foregoing embodiments describedabove), R_(b) is H. For example, embodiments described herein, in whichR₃ is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, heteroaryl, or —O—C₁-C₆alkylene-phenyl, can include one or more of the following features: R₁is H; R₂ is H; X is —C(O)NR^(c)R^(d) (e.g., —C(O)NH₂) or heteroaryl(e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-triazolyl (optionallysubstituted with from 1-2 independently selected C₁-C₆ alkyl), pyridyl,or pyrimidinyl)); R is H or —C(O)—C₁-C₆alkyl (e.g., —C(O)—CH₃); R₅ isC₁-C₆ alkyl (e.g., —CH₃); R₄ is H; R_(b) is H. As another example,compounds of formula (II) can include one or more of the followingfeatures: R₁ is H; R₂ is H; X is —C(O)NR^(c)R^(d) (e.g., —C(O)NH₂) orheteroaryl (e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-triazolyl(optionally substituted with from 1-2 independently selected C₁-C₆alkyl), pyridyl, or pyrimidinyl)); R is H or —C(O)—C₁-C₆alkyl (e.g.,—C(O)—CH₃); R₅ is C₁-C₆ alkyl (e.g., —CH₃); R₄ is H; R_(b) is H.

In some embodiments, a disclosed compound includes those delineated inTable 1 and/or the Examples, e.g., one having the formula:

The compounds of the present disclosure and formulations thereof mayhave a plurality of chiral centers. Each chiral center may beindependently R, S, or any mixture of R and S. For example, in someembodiments, a chiral center may have an R:S ratio of between about100:0 and about 50:50, between about 100:0 and about 75:25, betweenabout 100:0 and about 85:15, between about 100:0 and about 90:10,between about 100:0 and about 95:5, between about 100:0 and about 98:2,between about 100:0 and about 99:1, between about 0:100 and 50:50,between about 0:100 and about 25:75, between about 0:100 and about15:85, between about 0:100 and about 10:90, between about 0:100 andabout 5:95, between about 0:100 and about 2:98, between about 0:100 andabout 1:99, between about 75:25 and 25:75, and about 50:50. Formulationsof the disclosed compounds comprising a greater ratio of one or moreisomers (i.e., R and/or S) may possess enhanced therapeuticcharacteristic relative to racemic formulations of a disclosed compoundsor mixture of compounds. In some instances, chemical formulas containthe descriptor “—(R)—” or “—(S)—” that is further attached to solidwedge or dashed wedge. This descriptor is intended to show a methinecarbon (CH) that is attached to three other substituents and has eitherthe indicated R or S configuration (see, e.g., Table 1).

Disclosed compounds may provide for efficient cation channel opening atthe NMDA receptor, e.g. may bind or associate with the glutamate site ofthe NMDA receptor to assist in opening the cation channel. The disclosedcompounds may be used to regulate (turn on or turn off) the NMDAreceptor through action as an agonist.

The compounds as described herein may be glycine site NMDA receptorpartial agonists. A partial agonist as used in this context will beunderstood to mean that at a low concentration, the analog acts as anagonist and at a high concentration, the analog acts as an antagonist.Glycine binding is not inhibited by glutamate or by competitiveinhibitors of glutamate, and also does not bind at the same site asglutamate on the NMDA receptor. A second and separate binding site forglycine exists at the NMDA receptor. The ligand-gated ion channel of theNMDA receptor is, thus, under the control of at least these two distinctallosteric sites. Disclosed compounds may be capable of binding orassociating with the glycine binding site of the NMDA receptor. In someembodiments, disclosed compounds may possess a potency that is 10-foldor greater than the activity of existing NMDA receptor glycine sitepartial agonists.

The disclosed compounds may exhibit a high therapeutic index. Thetherapeutic index, as used herein, refers to the ratio of the dose thatproduces a toxicity in 50% of the population (i.e., TD₅₀) to the minimumeffective dose for 50% of the population (i.e., ED₅₀) Thus, thetherapeutic index=(TD₅₀):(ED₅₀). In some embodiments, a disclosedcompound may have a therapeutic index of at least about 10:1, at leastabout 50:1, at least about 100:1, at least about 200:1, at least about500:1, or at least about 1000:1.

Compositions

In other aspects, formulations and compositions comprising the disclosedcompounds and optionally a pharmaceutically acceptable excipient areprovided. In some embodiments, a contemplated formulation comprises aracemic mixture of one or more of the disclosed compounds.

Contemplated formulations may be prepared in any of a variety of formsfor use. By way of example, and not limitation, the compounds may beprepared in a formulation suitable for oral administration, subcutaneousinjection, or other methods for administering an active agent to ananimal known in the pharmaceutical arts.

Amounts of a disclosed compound as described herein in a formulation mayvary according to factors such as the disease state, age, sex, andweight of the individual. Dosage regimens may be adjusted to provide theoptimum therapeutic response. For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for themammalian subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier.

The specification for the dosage unit forms of the invention aredictated by and directly dependent on (a) the unique characteristics ofthe compound selected and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, monostearate salts and gelatin.

The compounds can be administered in a time release formulation, forexample in a composition which includes a slow release polymer. Thecompounds can be prepared with carriers that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations aregenerally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating thecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In accordance with an alternative aspect of the invention, a compoundmay be formulated with one or more additional compounds that enhance thesolubility of the compound.

Methods

Methods for treating a condition in a patient in need thereof byadministering a therapeutically effective dose of a compound describedherein are provided. In some embodiments, the condition may be a mentalcondition. For example, a mental illness may be treated. In anotheraspect, a nervous system condition may be treated. For example, acondition that affects the central nervous system, the peripheralnervous system, and/or the eye may be treated. In some embodiments,neurodegenerative diseases may be treated.

In some embodiments, the methods include administering a compound totreat patients suffering from autism, anxiety, depression, bipolardisorder, attention deficit disorder, attention deficit hyperactivitydisorder (ADHD), schizophrenia, a psychotic disorder, a psychoticsymptom, social withdrawal, obsessive-compulsive disorder (OCD), phobia,post-traumatic stress syndrome, a behavior disorder, an impulse controldisorder, a substance abuse disorder (e.g., a withdrawal symptom, opiateaddiction, nicotine addiction, and ethanol addition), a sleep disorder,a memory disorder (e.g., a deficit, loss, or reduced ability to make newmemories), a learning disorder, urinary incontinence, multiple systematrophy, progressive supra-nuclear palsy, Friedrich's ataxia, Down'ssyndrome, fragile X syndrome, tuberous sclerosis,olivio-ponto-cerebellar atrophy, cerebral palsy, drug-induced opticneuritis, ischemic retinopathy, diabetic retinopathy, glaucoma,dementia, AIDS dementia, Alzheimer's disease, Huntington's chorea,spasticity, myoclonus, muscle spasm, Tourette's syndrome, epilepsy,cerebral ischemia, stroke, a brain tumor, traumatic brain injury,cardiac arrest, myelopathy, spinal cord injury, peripheral neuropathy,acute neuropathic pain, and chronic neuropathic pain.

In some embodiments, methods of treating a memory disorder associatedwith aging, schizophrenia, special learning disorders, seizures,post-stroke convulsions, brain ischemia, hypoglycemia, cardiac arrest,epilepsy, migraine, AIDS dementia, Huntington's chorea, Parkinson'sdisease, early stage Alzheimer's disease, and Alzheimer's disease arecontemplated.

In certain embodiments, methods for treating schizophrenia are provided.For example, paranoid type schizophrenia, disorganized typeschizophrenia (i.e., hebephrenic schizophrenia), catatonic typeschizophrenia, undifferentiated type schizophrenia, residual typeschizophrenia, post-schizophrenic depression, and simple schizophreniamay be treated using the methods and compositions contemplated herein.Psychotic disorders such as schizoaffective disorders, delusionaldisorders, brief psychotic disorders, shared psychotic disorders, andpsychotic disorders with delusions or hallucinations may also be treatedusing the compositions contemplated herein.

Paranoid schizophrenia may be characterized where delusions or auditoryhallucinations are present, but thought disorder, disorganized behavior,or affective flattening are not. Delusions may be persecutory and/orgrandiose, but in addition to these, other themes such as jealousy,religiosity, or somatization may also be present. Disorganized typeschizophrenia may be characterized where thought disorder and flataffect are present together. Catatonic type schizophrenia may becharacterized where the patient may be almost immobile or exhibitagitated, purposeless movement. Symptoms can include catatonic stuporand waxy flexibility. Undifferentiated type schizophrenia may becharacterized where psychotic symptoms are present but the criteria forparanoid, disorganized, or catatonic types have not been met. Residualtype schizophrenia may be characterized where positive symptoms arepresent at a low intensity only. Post-schizophrenic depression may becharacterized where a depressive episode arises in the aftermath of aschizophrenic illness where some low-level schizophrenic symptoms maystill be present. Simple schizophrenia may be characterized by insidiousand progressive development of prominent negative symptoms with nohistory of psychotic episodes.

In some embodiments, methods are provided for treating psychoticsymptoms that may be present in other mental disorders, including, butnot limited to, bipolar disorder, borderline personality disorder, drugintoxication, and drug-induced psychosis. In another embodiment, methodsfor treating delusions (e.g., “non-bizarre”) that may be present in, forexample, delusional disorder are provided.

Also provided are methods for treating social withdrawal in conditionsincluding, but not limited to, social anxiety disorder, avoidantpersonality disorder, and schizotypal personality disorder.

In some embodiments, methods are provided for treating neuropathic pain.The neuropathic pain may be acute or chronic. In some cases, theneuropathic pain may be associated with a condition such as herpes, HIV,traumatic nerve injury, stroke, post-ischemia, fibromyalgia, reflexsympathetic dystrophy, complex regional pain syndrome, spinal cordinjury, sciatica, phantom limb pain, diabetic neuropathy, and cancerchemotherapeutic-induced neuropathic pain. Methods for enhancing painrelief and for providing analgesia to a patient are also contemplated.

Further contemplated methods include a method of treating autism and/oran autism spectrum disorder in a patient need thereof, comprisingadministering an effective amount of a compound to the patient. In anembodiment, a method for reducing the symptoms of autism in a patient inneed thereof is contemplated, comprising administering an effectiveamount of a disclosed compound to the patient. For example, uponadministration, the compound may decrease the incidence of one or moresymptoms of autism such as eye contact avoidance, failure to socialize,attention deficit, poor mood, hyperactivity, abnormal sound sensitivity,inappropriate speech, disrupted sleep, and preservation. Such decreasedincidence may be measured relative to the incidence in the untreatedindividual or an untreated individual(s).

Also provided herein is a method of modulating an autism target geneexpression in a cell comprising contacting a cell with an effectiveamount of a compound described herein. The autism gene expression may befor example, selected from ABAT, APOE, CHRNA4, GABRA5, GFAP, GRIN2A,PDYN, and PENK. In another embodiment, a method of modulating synapticplasticity in a patient suffering from a synaptic plasticity relateddisorder is provided, comprising administering to the patient aneffective amount of a compound.

In another embodiment, a method of treating Alzheimer's disease, ore.g., treatment of memory loss that e.g., accompanies early stageAlzheimer's disease, in a patient in need thereof is provided,comprising administering a compound. Also provided herein is a method ofmodulating an Alzheimer's amyloid protein (e.g., beta amyloid peptide,e.g. the isoform Aβ₁₋₄₂), in-vitro or in-vivo (e.g. in a cell)comprising contacting the protein with an effective amount of a compoundis disclosed. For example, in some embodiments, a compound may block theability of such amyloid protein to inhibit long-term potentiation inhippocampal slices as well as apoptotic neuronal cell death. In someembodiments, a disclosed compound may provide neuroprotective propertiesto a Alzheimer's patient in need thereof, for example, may provide atherapeutic effect on later stage Alzheimer's associated neuronal celldeath.

In a further embodiment, a method of treating depression comprisingadministering a compound described herein is provided. In someembodiments, the treatment may relieve depression or a symptom ofdepression without affecting behavior or motor coordination and withoutinducing or promoting seizure activity. Exemplary depression conditionsthat are expected to be treated according to this aspect of theinvention include, but are not limited to, major depressive disorder,dysthymic disorder, psychotic depression, postpartum depression,premenstrual syndrome, premenstrual dysphoric disorder, seasonalaffective disorder (SAD), bipolar disorder (or manic depressivedisorder), mood disorder, and depressions caused by chronic medicalconditions such as cancer or chronic pain, chemotherapy, chronic stress,and post traumatic stress disorders. In addition, patients sufferingfrom any form of depression often experience anxiety. Various symptomsassociated with anxiety include fear, panic, heart palpitations,shortness of breath, fatigue, nausea, and headaches among others.Anxiety or any of the symptoms thereof may be treated by administering acompound as described herein.

Also provided herein are methods of treating a condition intreatment-resistant patients, e.g., patients suffering from a mental orcentral nervous system condition that does not, and/or has not,responded to adequate courses of at least one, or at least two, othercompounds or therapeutics. For example, provided herein is a method oftreating depression in a treatment resistant patient, comprising a)optionally identifying the patient as treatment resistant and b)administering an effective dose of a compound to said patient.

In some embodiments, a compound described herein may be used for acutecare of a patient. For example, a compound may be administered to apatient to treat a particular episode (e.g., a severe episode) of acondition contemplated herein.

Also contemplated herein are combination therapies comprising a compoundin combination with one or more other active agents. For example, acompound may be combined with one or more antidepressants, such astricyclic antidepressants, MAO-I's, SSRI's, and double and triple uptakeinhibitors and/or anxiolytic drugs. Exemplary drugs that may be used incombination with a compound include Anafranil, Adapin, Aventyl, Elavil,Norpramin, Pamelor, Pertofrane, Sincquan, Surmontil, Tofranil, Vivactil,Parnate, Nardil, Marplan, Celexa, Lexapro, Luvox, Paxil, Prozac, Zoloft,Wellbutrin, Effexor, Remeron, Cymbalta, Desyrel (trazodone), andLudiomill. In another example, a compound may be combined with anantipsychotic medication. Non-limiting examples of antipsychoticsinclude butyrophenones, phenothiazines, thioxanthenes, clozapine,olanzapine, risperidone, quetiapine, ziprasidonc, amisulpride,asenapine, paliperidone, iloperidone, zotepine, sertindole, lurasidone,and aripiprazole. It should be understood that combinations of acompound and one or more of the above therapeutics may be used fortreatment of any suitable condition and are not limited to use asantidepressants or antipsychotics.

EXAMPLES

The following examples are provided for illustrative purposes only, andare not intended to limit the scope of the disclosure.

Table 1 below shows some exemplary compounds of the disclosure andprovides physiochemical characteristics of the compounds.

TABLE 1 Molecular Weight Compound Structure (Da) cLogP tPSA Compound A

385 −4.42 179 Compound B

525 −0.79 169.8 Compound C

393 −3.47 163.7 Compound D

447 −2.61 141 Compound E

418 −2.66 159.6 Compound F

418 −3.36 159.6 Compound G

428 −2.14 146.4 Compound H

326 −3.44 133 Compound I

380 −2.59 110.3 Compound J

351 −2.63 128.9 Compound K

351 −3.33 159.6 Compound L

361 −1.88 115.7 6S-FNL-6

499.4388 −4.42387 179.29 6S-FNL-2

460.4803 −0.65328 148.34 6S-FNL-4

418.4436 −1.09441 142.27 6S-FNL-3

326.3483 −2.60699 130.83 6S-FNL-5

284.3116 −3.04811 124.76 6S-FNL-24

525.5569 −0.786001 169.82 6S-FNL-8

393.3977 −3.46511 163.75 6S-FNL-9

447.4882 −2.61196 140.97 6S-FNL-12

418.4072 −3.35707 159.58 6S-FNL-21

428.4451 −2.14114 146.44 6S-FNL-7- F1, 6S- FNL-7-F2

326.3483 −3.43817 133.04 6S-FNL-10

380.4387 −2.58502 110.26 6S-FNL-16

351.3577 −2.63355 128.87 6S-FNL-14

351.3577 −3.33013 128.87 6S-FNL-19

361.3956 −1.88186 115.73 6S-FNL-25

543.5474 −0.643299 169.82 6S-FNL-26

555.5829 −0.943672 179.05 6S-FNL-27

539.5835 −0.27258 169.82 6S-FNL-28

543.5474 −0.643299 169.82 6S-FNL-29

543.5474 −0.643299 169.82 6S-FNL-30

531.6046 −0.396191 169.82 6S-FNL-31

517.578 −0.84076 169.82 6S-FNL-23

384.4274 −1.76492 142.27 6S-FNL-22

354.4014 −2.19465 133.04 6S-FNL-18

409.4369 −0.960294 138.1 6S-FNL-17

379.4109 −1.39002 128.87 6S-FNL-11

309.3211 −2.94007 120.59 6S-FNL-15

409.4369 −1.65688 138.1 6S-FNL-13

379.4109 −2.08661 128.87 6S-FNL-20

419.4748 −0.35934 124.96 6S-1

485.5313 −2.93177 191.6 6S-3

338.402 −2.19496 101.98

Example 1—Synthesis of Compound A

Synthesis of 1, 3, 5-Tris (4-methoxybenzyl)-1, 3, 5-triazinane (1)

To a stirring solution of SM-1 (200 g, 1.46 mol) in EtOH (600 mL) atroom temperature was added formaldehyde (33% aq, 105 mL) drop wise. Thereaction mixture was stirred at room temperature for 1 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with EtOAc (100 mL) and washed with water (100 mL) followed bybrine. The separated organic layer was concentrated under reducedpressure to obtained crude; which was triturated with n-hexane to afford1 (200 g, 30.6%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.18 (d, J=8.0 Hz, 6H), 6.81 (d, J=8.0 Hz,6H), 3.71 (s, 9H), 3.50 (s, 6H), 3.29 (s, 6H).

Synthesis of Benzyl 2-(4-methoxybenzyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (2)

To a stirring solution of 1 (45 g, 100 mmol) in CH₂Cl₂ (150 mL) wasadded BF₃.OEt₂ (37 mL, 301 mmol) drop wise at −40° C. The above mixturewas added to a stirring solution of Int-B (95 g, 342 mmol) and Et₃N(210.2 mL, 1.50 mol) in dry CH₂Cl₂ (500 mL) drop wise. The reactionmixture was stirred at −40° C. for 45 min. The resulting reactionmixture was allowed to warm to RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was washed withsaturated NaHCO₃ solution (1×150 mL) followed by brine. The separatedorganic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The crude material was dissolved in EtOAc and kept inthe refrigerator for crystallization. Obtained crystals were filteredand washed with cold EtOAc (50 mL) and dried under vacuum to afford 2(90 g, 65%) as white crystalline solid.

¹H-NMR: (500 MHz, DMSO-d6): 7.36-7.30 (m, 5H), 7.24 (d, J=8.0 Hz, 1H),7.06 (d, J=8.0 Hz, 1H), 6.90 (d, J=7.5 Hz, 1H), 6.81 (d, J=8.5 Hz, 1H),5.09 (s, 2H), 4.29 (s, 1H), 4.13, 3.96 (dd, J=15.5 Hz, 15.0 Hz, 1H),3.73 (s, 3H), 3.11 (t, J=5.0 Hz, 2H), 2.16-2.09 (m, 2H), 1.83-1.77 (m,2H), 1.20-1.15 (m, 2H).

LCMS m/z: 381 [M⁺1]

Synthesis of Benzyl 1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate (3)

To a stirring solution of 2 (46 g, 121 mmol) in MeCN (460 mL) and H₂O(200 mL) were cooled to 0° C. and added a solution of CAN (199 g, 0.23mol) in H₂O (460 mL). The reaction mixture was stirred at roomtemperature for 1 h. The resulting mass was poured into ice cold water(100 mL) and the aqueous layer was extracted with EtOAc (2×200 mL). Thecombined organic layers were washed with saturated NaHCO₃ (1×150 mL)followed by brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford crude material waspurified by silica gel column chromatography eluting with EtOAc toobtained 3 (12 g, 38%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.90 (s, 1H), 7.36-7.29 (m, 5H), 5.10 (s,2H), 3.53 (d, J=4.5 Hz, 2H), 3.36-3.30 (m, 1H), 3.17, 3.13 (dd, J=5.0Hz, 5.0 Hz, 1H), 2.17-2.10 (m, 2H), 1.82-1.76 (m, 2H).

LCMS m/z: 261 [M⁺+1]

Synthesis of Benzyl 2-(2-ethoxy-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (4)

To a stirred solution of 3 (12 g, 46.1 mmol) in acetonitrile (120 mL)was added Cs₂CO₃ (37.6 g, 115.2 mmol) and ethyl 2-bromoacetate (7.7 mL,69.2 mmol) at RT and stirred for 16 h. After completion of reaction (byTLC), the volatiles were evaporated under reduced pressure. The residuewas diluted with water (50 mL) and extracted with EtOAc (2×100 mL). Theseparated organic layer was washed with brine, dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The obtained crudematerial was purified by silica gel column chromatography eluting with80% EtOAc/hexane to afford 4 (12.5 g, 78.6%) as pale brown syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.35-7.30 (m, 5H), 5.06 (s, 2H), 4.21 (s,1H), 4.18 (s, 1H), 4.13-4.10 (m, 2H), 3.69 (d, J=4.5 Hz, 1H), 3.47-3.44(m, 3H), 2.16 (t, J=6.0 Hz, 2H), 1.87-1.80 (m, 2H), 1.21-1.14 (m, 3H).

LCMS m/z: 369.3 [M⁺+Na]

Synthesis of 2-(5-((benzyloxy) carbonyl)-1-oxo-2, 5-diazaspiro [3.4]octan-2-yl) acetic acid (5)

To a stirred solution of 4 (9.0 g, 26.0 mmol) in THF/H₂O (80 mL/30 mL)was added LiOH.H₂O (2.73 g, 65.0 mmol) at RT and stirred for 3 h. Afterconsumption of the starting material (by TLC), the volatiles wereevaporated under reduced pressure. The residue was diluted with water(25 mL), extracted with EtOAc (2×50 mL). The separated aqueous layer wasacidified to pH˜3 using 2N HCl and extracted with EtOAc (3×50 mL). Theorganic layers were dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to afford 5 (7.0 g, 85.3%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.5 (br s, 1H), 7.35-7.30 (m, 5H), 5.06(s, 2H), 4.21 (s, 1H), 4.18 (s, 1H), 3.69 (d, J=4.5 Hz, 1H), 3.47-3.44(m, 3H), 2.16 (t, J=6.0 Hz, 2H), 1.87-1.80 (m, 2H)

Synthesis of benzyl2-(2-(((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)amino)-2-oxoethyl)-1-oxo-2,5-diazaspiro [3.4] octane-5-carboxylate (6)

To a stirring solution of 5 (4 g, 12.5 mmol) in DCM (50 mL) were addedN, N-diisopropylethylamine (5.79 mL, 31.2 mmol), Int-E (1.78 g, 15.0mmol), followed by HATU (7.16 g, 18.7 mmol) at 0° C. and stirred at RTfor 16 h. After consumption of the starting material (by TLC), thereaction mixture was diluted with water (20 mL), the organic layer waswashed with citric acid solution (1×75 mL) followed by brine solution(1×50 mL). The separated organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure to afford crude which waspurified by column chromatography to obtained 6 (2.1 g, 40.3%) asoff-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.80-7.73 (m, 1H), 7.38-7.31 (m, 5H), 7.23(d, J=10.5 Hz, 1H), 7.08 (d, J=8.0 Hz, 1H), 5.08-5.01 (m, 1H), 4.86 (t,J=4.0 Hz, 1H), 4.12-4.00 (m, 1H), 3.88-3.83 (m, 1H), 3.70-3.67 (m, 2H),3.60-3.51 (m, 3H), 2.18-2.11 (m, 2H), 1.33-1.22 (m, 4H), 1.00 (d, J=6.5Hz, 3H)

Synthesis of (2S, 3R)-3-hydroxy-2-(2-(1-oxo-2, 5-diazaspiro [3.4]octan-2-yl)acetamido)butanamide (7)

To a stirring solution of 6 (2.1 g, 5.02 mmol) in MeOH (20 mL) was added(50% wet) 10% Pd/C (1.0 g) and stirred under H₂ atmosphere (balloonpressure) at RT for 2 h. After completion of reaction (by TLC), thereaction mixture was filtered through a pad of celite using EtOAc/MeOH(10 mL/10 mL). The filtrate was concentrated under reduced pressure toafford 7 (1.4 g, 98.5%) as off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.75 (d, J=8.0 Hz, 1H), 7.24 (s, 2H),4.08-4.06 (m, 2H), 4.01-3.95 (m, 2H), 3.93-3.84 (m, 1H), 3.37-3.34 (m,2H), 3.33 (d, J=4.5 Hz, 1H), 2.90-2.87 (m, 2H), 1.90 (t, J=7.0 Hz, 2H),1.77-1.66 (m, 2H), 1.01 (d, J=6.5 Hz, 3H)

LCMS m/z: 285.3 [M⁺+1]

Synthesis of tert-butyl ((2S, 3R)-1-(2-(2-(((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)amino)-2-oxoethyl)-1-oxo-2,5-diazaspiro [3.4] octan-5-yl)-3-hydroxy-1-oxobutan-2-yl) carbamate (8)

To a stirring solution of 7 (300 mg, 1.05 mmol) in DCM (25 mL), DMF (0.5mL) were added N, N-diisopropylethylamine (0.58 mL, 3.15 mmol), Int-C(277 mg, 1.26 mmol), followed by HATU (481 mg, 1.26 mmol) at 0° C. andstirred at RT for 16 h. After consumption of the starting material (byTLC), the reaction mixture was evaporated under reduced pressure and theobtained crude was purified by column chromatography by eluting 6%MeOH/DCM to afford 8 (400 mg, 70% pure by LCMS) which was furtherpurified by preparative HPLC to yield pure 8 (230 mg, LCMS purity 96%)as off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.83 (d, J=8.5 Hz, 1H), 7.25 (s, 2H), 6.33(d, J=9.0 Hz, 1H), 4.80-4.75 (m, 2H), 4.24-3.98 (m, 4H), 3.71-3.60 (m,3H), 3.39-3.33 (m, 1H), 2.14-1.89 (m, 4H), 1.41 (s, 9H), 1.14-1.05 (m,6H)

LCMS m/z: 486.3 [M⁺+1]

Synthesis of (2S, 3R)-2-(2-(5-((2S,3R)-2-amino-3-hydroxybutanoyl)-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl)acetamido)-3-hydroxybutanamide (Compound A)

To a stirring solution of 8 (130 mg, 0.26 mmol) in DCM (3 mL) was addedTFA (152 mg, 1.34 mmol) at 0° C. and stirred at RT for 2 h. Aftercompletion of starting material (by TLC), the reaction mixture wasconcentrated under reduced pressure and co-distilled with DCM to affordCompound A (110 mg, 82.7% LCMS purity 93.32%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 4.33-4.28 (m, 1H), 4.23-4.19 (m, 4H),4.05-4.01 (m, 1H), 3.91-3.87 (m, 1H), 3.71-3.65 (m, 2H), 3.55-3.49 (m,1H), 2.30-2.24 (m, 2H), 2.02-1.97 (m, 2H), 1.27 (t, J=4.0 Hz, 3H), 1.15(t, J=5.5 Hz, 3H)

LCMS m/z: 386.3 [M⁺+1]

Synthesis of (S)-1-((benzyloxy) carbonyl) pyrrolidine-2-carboxylic acid(Int A)

To a stirring solution of L-proline (250 g, 2.17 mol) in water (1 L) wasadded Na₂CO₃ (576 g, 5.43 mol) and stirred for 1 h. After being cooledto 0° C., benzylchloroformate (50% in PhCH₃) (444 g, 2.61 mol) was addeddrop wise to the reaction mixture and again stirred for 1 h. Theresulting reaction mixture was warmed to RT and further stirred for 24h. After consumption of the starting material (by TLC), the reaction wasdiluted with water (1 L) and ether (1.5 L). The separated aqueous layerwas treated with PhCH₃ (1.5 L) and acidified with 6N HCl. The aqueouslayer was extracted with EtOAc (3×1.5 L), combined organic extracts werewashed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford Int A (450 g, 84%) as paleyellow syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.71 (br s, 1H), 7.37-7.26 (m, 5H),5.07-4.99 (m, 2H), 4.25-4.15 (m, 1H), 3.45-3.34 (m, 2H), 2.25-2.14 (m,1H), 1.94-1.79 (m, 3H)

LCMS m/z: 250.4 [M⁺+1]

Synthesis of (S)-benzyl 2-(chlorocarbonyl) pyrrolidine-1-carboxylate(Int-B)

To a stirring solution of Int-A (2.5 g, 0.01 mol) in CH₂Cl₂ (50 mL) wasadded SOCl₂ (2.7 g, 0.02 mol) at 0° C. and stirred for 2 h. The reactionmixture was concentrated under reduced pressure to afford Int-B ascrude. This material was directly used for the next step without furtherpurification.

Synthesis of (2S, 3R)-2-((tert-butoxycarbonyl) amino)-3-hydroxybutanoicacid (Int-C)

To a stirring solution of (2S, 3R)-2-amino-3-hydroxybutanoic acid (SM-2)(30 g, 0.25 mol) in THF (150 mL) and water (150 mL) was added NaHCO₃ (65g, 0.75 mol) followed by Boc-anhydride (66 mL, 0.302 mol) at 0° C. Thereaction mixture was stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was extracted withEtOAc (2×150 mL). The aqueous layer was acidified using 2N HCl and thenextracted with 10% MeOH/CH₂Cl₂. The separated organic extracts weredried over anhydrous Na₂SO₄, filtered and concentrated under vacuum toafford Int-C (30 g, 63%).

¹H-NMR: (400 MHz, CDCl₃): δ 5.92-5.70 (m, 2H), 5.55 (d, 1H), 4.42 (br s,1H), 4.29 (d, 1H), 1.47 (s, 9H), 1.25 (d, 3H)

LCMS m/z: 218 [M⁺−1]

Synthesis of (2S, 3R)-methyl 2-amino-3-hydroxybutanoate (Int-D)

To a stirring solution of L-threonine (200 g, 1.68 mol) in methanol (1.2L) was added SOCl₂ (244 mL, 3.36 mol) drop wise at 0° C. and stirred for1 h. The resulting reaction mixture was refluxed for 24 h. Afterconsumption of the starting material (by TLC), the reaction mixture waswarmed to RT and concentrated under vacuum and decanted with n-hexane(2×50 mL). The residue was dissolved in EtOH (1 L) and neutralized withEt₃N (471 mL, 3.36 mol) and again stirred for 2 h. The precipitatedsolid was filtered off; obtained filtrate was concentrated under vacuumto afford Int-D (195 g, 80%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.51 (br s, 3H), 4.13-4.10 (m, 1H), 3.91(br s, 1H), 1.20 (d, 3H)

LCMS m/z: 134.1 [M⁺1]

Synthesis of (2S, 3R)-2-amino-3-hydroxybutanamide (Int-E)

A solution of Int-D (190 g, 1.35 mol) in IPA (2 L) was taken inautoclave and purged NH₃ gas (7-8 kg) and stirred at 35° C. for 24 h.Then removed NH₃ gas and reaction mixture was concentrated under reducedpressure and added CH₂Cl₂ and filtered. Obtained solid was refluxed inEtOH for 1 h at 78° C. The reaction mass was filtered in heatingcondition and n-hexane was added to the filtrate and again stirred foranother 4 h. Obtained precipitated solid was filtered and dried undervacuum to afford Int-E (160 g, 47%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.38 (br s, 1H), 7.02 (br s, 1H), 4.66 (brs, 1H), 3.77-3.70 (m, 1H), 2.93 (d, 1H), 2.72 (br m, 1H), 1.05 (d, 3H)

LCMS m/z: 119.1 [M⁺+1]

UPLC (ELSD purity): 99.9%

Example 2—Synthesis of Compound B

Synthesis of 1, 3, 5-Tris (4-methoxybenzyl)-1, 3, 5-triazinane (1)

To a stirring solution of (4-methoxyphenyl) methanamine SM-1 (200 g,1.46 mol) in EtOH (600 mL) at room temperature was added formaldehyde(33% aq, 105 mL) drop wise. The reaction mixture was stirred at roomtemperature for 1 h. After consumption of the starting material (byTLC), the reaction mixture was diluted with EtOAc (100 mL) and washedwith water (100 mL) followed by brine. The separated organic layer wasconcentrated under reduced pressure to obtain crude; which was washedwith n-hexane to afford 1 (200 g, 30.6%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.18 (d, J=8.0 Hz, 6H), 6.81 (d, J=8.0 Hz,6H), 3.71 (s, 9H), 3.50 (s, 6H), 3.29 (s, 6H)

Synthesis of Benzyl 2-(4-methoxybenzyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (2)

A stirring solution of Int-B (100 g, 0.37 mol) in dry CH₂Cl₂ (500 mL)was cooled to −40° C. and Et₃N (210.2 mL, 1.50 mol) was added drop wise.The reaction mixture was stirred at −40° C. for 45 min. To this amixture of 1 (50 g, 0.12 mol) and BF₃OEt₂ (47.6 g, 0.33 mol) in CH₂Cl₂(150 mL) was added drop wise at −40° C. The resulting reaction mixturewas allowed to stir at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was washed with saturated NaHCO₃solution followed by brine. The separated organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The crudematerial was dissolved in EtOAc and kept in the refrigerator forcrystallization. Obtained crystals were filtered and washed with coldEtOAc and dried under vacuum to afford 2 (82 g, 58%) as whitecrystalline solid.

¹H-NMR: (500 MHz, DMSO-d6): δ 7.36-7.30 (m, 5H), 7.24 (d, 0.1=8.0 Hz,1H), 7.06 (d, 8.0 Hz, 1H), 6.90 (d, J=7.5 Hz, 1H), 6.81 (d, J=8.5 Hz,1H), 5.09 (s, 2H), 4.29 (s, 1H), 4.13, 3.96 (dd, J=15.5 Hz, 15.0 Hz,1H), 3.73 (s, 3H), 3.11 (t, J=5.0 Hz, 2H), 2.16-2.09 (m, 2H), 1.83-1.77(m, 2H), 1.20-1.15 (m, 2H)

LCMS m/z: 381 [M⁺+1]

Synthesis of Benzyl 1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate (3)

To a stirring solution of 2 (30 g, 78.94 mmol) in MeCN (300 mL) and H₂O(150 mL) were cooled to 0° C. and added a solution of CAN (129 g, 0.23mol) in H₂O (300 mL). The reaction mixture was stirred at roomtemperature for 1 h. The resulting mass was poured into ice cold waterand the aqueous layer was extracted with EtOAc (2×150 mL). The combinedorganic layers were washed with saturated NaHCO₃ followed by brine,dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to get crude. Obtained material was purified by silica gelcolumn chromatography eluting with EtOAc to afford 3 (8 g, 40%) as anoff-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.90 (s, 1H), 7.36-7.29 (m, 5H), 5.10 (s,2H), 3.53 (d, J=4.5 Hz, 2H), 3.36-3.30 (m, 1H), 3.17, 3.13 (dd, J=5.0Hz, 5.0 Hz, 1H), 2.17-2.10 (m, 2H), 1.82-1.76 (m, 2H)

LCMS m/z: 261 [M⁺+1]

Synthesis of Benzyl 2-(2-ethoxy-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (4)

To a stirred solution of 3 (10.0 g, 38.46 mmol) in acetonitrile (100 mL)were added Cs₂CO₃ (31.34 g, 96.19 mmol) and ethyl 2-bromoacetate (6.42mL, 57.60 mmol) at RT and stirred for 16 h at RT. The volatiles wereevaporated under reduced pressure. The residue was diluted with waterand extracted with EtOAc (2×100 mL). The separated organic layer waswashed with brine, dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The obtained crude material was purified by silica gelcolumn chromatography eluting with 80% EtOAc/Hexane to afford 4 (10.0 g,75%) as pale brown syrup.

¹H-NMR: (500 MHz, DMSO-d6): δ 7.35-7.30 (m, 5H), 5.06 (s, 2H), 4.21 (s,1H), 4.18 (s, 1H), 4.13-4.10 (m, 2H), 3.69 (d, J=4.5 Hz, 1H), 3.47-3.44(m, 3H), 2.16 (t, J=6.0 Hz, 2H), 1.87-1.80 (m, 2H), 1.21-1.14 (m, 3H)

LCMS m/z: 261 [M⁺+1]

Synthesis of 2-(5-((benzyloxy) carbonyl)-1-oxo-2, 5-diazaspiro [3.4]octan-2-yl) acetic acid (5)

To a stirred solution of 4 (6.0 g, 17.34 mmol) in THF: H₂O (75 mL/40 mL)was added LiOH.H₂O (1.82 g, 43.33 mmol) at RT and stirred for 2 h. Afterconsumption of the starting material (by TLC), the volatiles wereevaporated under reduced pressure. The residue was diluted with water,washed with ether, the aqueous layer was acidified to pH˜2 using 2N HCland extracted with EtOAc (2×50 mL). The organic layers were washed withbrine, dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to afford 5 (4.5 g, 88.2%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.5 (br s, 1H), 7.35-7.30 (m, 5H), 5.06(s, 2H), 4.21 (s, 1H), 4.18 (s, 1H), 3.69 (d, J=4.5 Hz, 1H), 3.47-3.44(m, 3H), 2.16 (t, J=6.0 Hz, 2H), 1.87-1.80 (m, 2H)

LCMS m/z: 319 [M⁺+1]

Synthesis of 2 benzyl 2-(2-(((2S, 3R)-3-acetoxy-1-amino-1-oxobutan-2-yl)amino)-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate (6)

To a stirring solution of 5 (10 g, 31.44 mmol) in DMF (120 mL) was addedN, N-diisopropylethyl amine (14.48 mL, 78.6 mmol), Int-F (5.95 g, 37.7mmol), followed by HATU (14.33 g, 37.7 mmol) at 0° C. and stirred at RTfor 16 h. After consumption of the starting material (by TLC), thereaction mixture was diluted with water and washed with saturated NaHCO₃solution. The separated organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to give crude product,which was purified by column chromatography by 1% MeOH/DCM to afford 6(5.5 g, 38.1%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.97-7.88 (m, 1H), 7.49-7.46 (m, 5H), 7.35(s, 1H), 7.17 (s, 1H), 5.11 (s, 2H), 5.08-5.02 (m, 1H), 4.41 (d, 0.1=9.0Hz, 1H), 4.21-4.11 (m, 1H), 3.87-3.79 (m, 4H), 3.13 (s, 1H), 2.18-2.21(m, 2H), 1.98 (s, 3H), 1.90-1.84 (m, 2H), 1.81-1.10 (m, 3H)

LCMS m/z: 483.5 [M⁺+Na]

Synthesis of (2R, 3S)-4-amino-4-oxo-3-(2-(1-oxo-2, 5-diazaspiro [3.4]octan-2-yl) acetamido) butan-2-yl acetate (7)

To a stirring solution of 6 (5.5 g, 11.95 mmol) in methanol (50 mL) wasadded 50% wet 10% Pd/C (2.0 g) and stirred under H₂ atmosphere (balloonpressure) for 4 h at RT. The reaction mixture was filtered through a padof celite and triturated with methanol (5 mL). The filtrate wasconcentrated under reduced pressure to give 7 (3.80 g, 97.6%) as anoff-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.97 (d, J=9.6 Hz, 1H), 7.49 (s, 1H), 7.16(s, 1H), 5.19-5.13 (m, 1H), 4.46-4.41 (m, 1H), 4.03-3.90 (m, 2H),3.39-3.30 (m, 3H), 2.91 (t, J=6.4 Hz, 2H), 1.97 (s, 3H), 1.78-1.70 (m,2H), 1.29-1.15 (m, 3H), 1.50-1.10 (m, 2H).

LCMS m/z: 327.3 [M⁺+1]

Synthesis of (2R, 3S)-4-amino-3-(2-(5-(1-benzyl-5-methyl-1H-1, 2,3-triazole-4-carbonyl)-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl)acetamido)-4-oxobutan-2-yl acetate (Compound B)

To a stirring solution of Int-H2 (4.0 g, 18.43 mmol) in CH₂Cl₂ (20 mL),DMF (0.1 mL) was added oxalyl chloride (3.34 mL, 36.86 mmol) at 0° C.The reaction mixture was warmed to RT and stirred for 2 h. The volatileswere evaporated under reduced pressure in presence of N₂ atmosphere toafford acid chloride. To a stirred solution of acid chloride in DCM (40mL) was added d 7 (3.8 g, 11.65 mmol), N, N-diisopropylethylamine (6.44mL, 37.04 mmol) at 0° C. The resulting reaction mixture was stirred atRT for 2 h. After consumption of the starting material (by TLC), thereaction mixture was diluted with water (30 mL) and extracted withCH₂Cl₂ (2×30 mL). Combined organic extracts were washed by NaHCO₃ (2×25mL). The separated organic extracts were dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to give crude product, which waspurified by silica gel column chromatography eluting with 2% MeOH/CH₂Cl₂to afford Compound B (2.6 g, 42.5%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.87, 7.89 (dd, J=8.5 Hz, J=8.5 Hz, 1H),7.47 (d, J=11.0 Hz, 2H), 7.38-7.31 (m, 2H), 7.17 (t, 3H), 5.63 (s, 2H),5.20-5.16 (m, 1H), 4.43-4.41 (m, 1H), 4.24-4.14 (m, 1H), 4.02 (t, J=7.0Hz, 1H), 3.84 (s, 3H), 3.38-3.33 (m, 1H), 2.20-2.14 (m, 2H), 1.97 (s,3H), 1.92-1.80 (m, 5H), 1.16 (t, J=7.5 Hz, 3H)

LCMS m/z: 526.6 [M⁺+1]; HPLC: 51.34% & 46.08% (enantiomers)

Synthesis of (S)-1-((benzyloxy) carbonyl) pyrrolidine-2-carboxylic acid(Int-A)

To a stirring solution of L-proline (250 g, 2.17 mol) in water (1 L) wasadded Na₂CO₃ (576 g, 5.43 mol) and stirred for 1 h. After being cooledto 0° C., benzylchloroformate (50% in PhCH₃) (444 g, 2.61 mol) was addeddrop wise to the reaction mixture and again stirred for 1 h. Theresulting reaction mixture was warmed to RT and further stirred for 24h. After consumption of the starting material (by TLC), the reaction wasdiluted with water (1 L) and ether (1.5 L). The separated aqueous layerwas treated with PhCH₃ (1.5 L) and acidified using 6N HCl. The aqueouslayer was extracted with EtOAc (3×1.5 L), combined organic extracts werewashed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford Int-A (450 g, 84%) aslight yellow syrup.

¹H-NMR: (400 MHz, DMSO-d6): δ 12.71 (br s, 1H), 7.37-7.26 (m, 5H),5.07-4.99 (m, 2H), 4.25-4.15 (m, 1H), 3.45-3.34 (m, 2H), 2.25-2.14 (m,1H), 1.94-1.79 (m, 3H).

LCMS m/z: 250.4 [M⁺+1]

Synthesis of (S)-benzyl 2-(chlorocarbonyl) pyrrolidine-1-carboxylate(Int-B)

To a stirring solution of Int-A (2.5 g, 0.01 mol) in CH₂Cl₂ (50 mL) wasadded SOCl₂ (2.7 g, 0.02 mol) at 0° C. and stirred for 2 h. The reactionmixture was concentrated under reduced pressure to afford Int-B ascrude. This material was directly used for the next step without furtherpurification.

Synthesis of (2S, 3R)-2-(((benzyloxy) carbonyl) amino)-3-hydroxybutanoicacid (Int-C)

To a stirring solution of NaHCO₃ (529 g, 6.30 mol) in water (1 L) wasadded L-threonine (SM-2) (250 g, 2.10 mol) at RT and stirred for 30 min.The reaction mixture was cooled to 0° C. Cbz-Cl (850 mL, 2.52 mol, 50%in PhCH₃) was added drop wise and stirred for 1 h. The reaction mixturewas warmed to RT and again stirred for 28 h. To this MTBE (1 L) wasadded and stirred for 20 min. Separated aqueous layer in toluene wasstirred for 20 min. Aqueous layer was acidified with 1N HCl (pH˜1-2) andextracted with EtOAc (3×1.5 L). The organic layer was washed with brine,dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Thecrude material was stirred with dicyclohexylamine (819 mL, 4.20 mol) for4 h to get white solid, filtered and dried. Obtained solid was refluxedwith EtOAc (1.5 L) for 1 h and then filtered. The solid material wasdissolved in water (1 L) and acidified with dil.H₂SO₄ and again stirredfor 30 min. The aqueous layer was extracted with EtOAc (3×1 L). Theseparated organic layer was washed with brine, dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure. Obtained crudematerial was triturated with n-hexane to afford Int-C (230 g, 43%) aswhite solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.55 (br m, 1H), 7.37-7.30 (m, 5H), 6.94(d, J=8.8 Hz, 1H), 5.05 (s, 2H), 4.08-3.94 (m, 2H), 1.02 (d, J=6.4 Hz,3H)

ELSD purity: 84.66%

Synthesis of benzyl ((2S, 3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)carbamate (Int-D)

To a solution of Int-C (25 g, 98.8 mmol) in DCM (250 mL) was addedammonium chloride (7.86 g, 147 mmol), HATU (45 g, 118 mmol), N,N-diisopropylethyl amine (45.5 mL, 261 mmol) and stirred at RT for 16 h.After completion of starting material (by TLC), the organic layer waswashed by saturated sodium bicarbonate solution (1×150 mL) followed by2N HCl (1×100 mL). After the separated organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure.Obtained crude material was purified by column chromatography by elutingwith 2% MeOH/DCM to afford Int-D (16 g, 66%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.36-7.32 (m, 5H), 7.04 (s, 1H), 7.25 (s,1H), 5.03 (s, 2H), 4.75 (d, 0.1=6.0 Hz, 1H), 3.95-3.92 (m, 1H),3.86-3.83 (m, 1H), 1.27-1.23 (m, 1H), 1.04 (d, J=6.5 Hz, 3H)

LCMS m/z: 253.3 [M⁺+1]

Synthesis of (2R, 3S)-4-amino-3-(((benzyloxy) carbonyl)amino)-4-oxobutan-2-yl acetate (Int-E)

To a stirring solution of Int-D (16 g, 63.4 mmol) in CH₂Cl₂ (250 mL)were added Et₃N (10.5 mL, 76.0 mmol) and DMAP (773 mg, 6.34 mmol), Ac₂O(7.12 mL, 76.0 mmol) at 0° C. and stirred at RT for 2 h. Aftercompletion of starting material (by TLC), the organic layer was washedwith water (1×150 mL) followed by brine (1×100 mL) washing. After theseparated organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. Obtained crude material waspurified by column chromatography by eluting with 1% MeOH/DCM to affordInt-E (15 g, 80.3%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.45 (s, 1H), 7.35-7.30 (m, 5H), 7.24 (d,J=9.2 Hz, 1H), 7.17 (s, 1H), 5.09-5.05 (m, 1H), 5.01 (s, 2H), 4.14-4.10(m, 1H), 1.93 (s, 3H), 1.14 (d, J=6.4 Hz, 3H)

LCMS m/z: 295.1 [M⁺+1]

Synthesis of (2R, 3S)-3, 4-diamino-4-oxobutan-2-yl acetate (Int-F)

To a stirring solution of Int-E (15 g, 51 mmol) in methanol (500 mL) wasadded 50% wet 10% Pd/C (4 g) and stirred under H₂ atmosphere (balloonpressure) at RT for 4 h. The reaction mixture was filtered through a padof celite and triturated with methanol (50 mL). The filtrate wasconcentrated under reduced pressure to afford Int-F (7.5 g, 91.9%) as anoff-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.59 (d, J=8.8 Hz, 1H), 7.16 (s, 1H), 7.01(s, 1H), 4.78 (d, J=5.2 Hz, 1H), 4.10 (m, 1H), 4.00-3.96 (m, 1H), 1.89(s, 3H), 1.01 (d, J=6.4 Hz, 3H).

LCMS m/z: 161.5 [M⁺+1]

Synthesis of (Azidomethyl) Benzene (SM-4)

To a stirring solution of benzyl bromide (30 g, 175 mmol) in dimethylformamide (300 mL) was added sodium azide (45.6 g, 701 mmol) at RT underinert atmosphere. The resultant reaction mixture was stirred at 70° C.for 16 h. After completion of reaction (monitored by TLC), the reactionmixture was allowed to RT; the volatiles were diluted with water (300mL) and ether (200 mL). The separated organic layer was washed by (3×200mL) of chilled water. The separated organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toafford compound SM-4 (18 g, crude) as an off-white solid.

¹H-NMR: (400 MHz, CDCl₃): δ 7.40-7.29 (m, 5H), 4.32 (s, 2H).

Synthesis of ethyl 1-benzyl-5-methyl-1H-1, 2, 3-triazole-4-carboxylate(Int-G1 & Int-G2)

To a stirring solution of SM-3 (8.0 g, 71.3 mmol) in toluene (80 mL) wasadded SM4 (12.0 g, 107 mmol) at RT under inert atmosphere. The resultantreaction mixture was heated to 100° C. and stirred for 16 h. Thereaction mixture was allowed to RT; the volatiles were evaporated underreduced pressure to which, crude residue was purified by columnchromatography by eluting 40% EtOAc/hexane to afford two isomers Int-G1(8 g) & Int-G2 (8.2 g).

¹H-NMR (Int-G1): (400 MHz, CDCl₃): δ 7.30-7.26 (m, 5H), 5.86 (s, 2H),4.34 (q, J=7.2 Hz, 2H), 2.53 (s, 3H), 1.33 (t, J=7.2 Hz, 3H)

LCMS m/z: 246.3 [M⁺+1].

¹H-NMR (Int-G2): (400 MHz, CDCl₃): δ 7.36-7.31 (m, 3H), 7.16 (t, J=6.0Hz, 2H), 5.53 (s, 2H), 4.43 (q, J=7.2 Hz, 2H), 2.45 (s, 3H), 1.41 (t,J=7.2 Hz, 3H)

LCMS m/z: 246.3 [M⁺+1]

Synthesis of 1-benzyl-5-methyl-1H-1, 2, 3-triazole-4-carboxylic acid(Int-H2)

To a stirring solution of compound Int-G2 (8.2 g, 33.4 mmol) in THF/H₂O(82 mL/82 mL, 1:1) was added LiOH.H₂O (4.2 g, 0.40 mmol) at RT andstirred for 16 h. After completion of reaction (by TLC), the volatileswere evaporated under reduced pressure. The residue was acidified withaqueous 2N HCl and the precipitated solid was filtered and washed withwater (25 mL), dried under reduced pressure to afford compound Int-H2(7.0 g, 97.2%) as an off-white solid.

¹H-NMR (H₂): (400 MHz, DMSO-d₆): δ 13.01 (br s, 1H), 7.40-7.32 (m, 5H),5.63 (s, 2H), 2.45 (s, 3H).

LCMS m/z: 218.3[M⁺+1].

Example 3—Synthesis of Compound C

Synthesis of 1, 3, 5-Tris (4-methoxybenzyl)-1, 3, 5-triazinane (1)

To a stirring solution of (4-methoxyphenyl) methanamine SM (200 g, 1.46mol) in EtOH (600 mL) at room temperature was added formaldehyde (33%aq, 105 mL) drop wise. The reaction mixture was stirred at roomtemperature for 1 h. After consumption of the starting material (asdetermined by TLC), the reaction mixture was diluted with EtOAc (100 mL)and washed with water (100 mL) followed by brine. The separated organiclayer was concentrated under reduced pressure to obtain crude; which wasfinally washed with n-hexane to afford 1 (200 g, 30.6%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.18 (d, 6H), 6.84 (d, 6H), 3.73 (s, 9H),3.50 (s, 6H), 3.29 (s, 6H).

Synthesis of Benzyl 2-(chlorocarbonyl)pyrrolidine-1-carboxylate (Int-B)

To a stirring solution of Int-A (100 g, 0.40 mol) in CH₂Cl₂ (500 mL) wasadded catalytic amount of DMF (1 mL) and the reaction mixture was cooledto 0° C. To this oxalyl chloride (112.3 mL, 0.60 mol) was added dropwise and the reaction mixture was stirred at room temperature for 2 h.After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure to afford Int-B (100 g)as crude. This material was directly used for the next step withoutfurther purification.

Synthesis of Benzyl 2-(4-methoxybenzyl)-1-oxo-2,5-diazaspiro [3.4]octane-5-carboxylate (2)

To a stirring solution of Int-B (100 g, 0.37 mol) in dry CH₂Cl₂ (500 mL)was cooled to −40° C. and added Et₃N (210.2 mL, 1.50 mol) drop wise. Thereaction mixture was stirred at −40° C. for 45 min. To this, a mixtureof compound 1 (50 g, 0.12 mol) in CH₂Cl₂ (150 mL) and BF₃.OEt₂ (47.6 g,0.33 mol) was added drop wise at −40° C. The resulting reaction mixturewas allowed to stir at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was washed with saturated NaHCO₃solution followed by brine. The separated organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The crudematerial was dissolved in EtOAc and kept in the refrigerator forcrystallization. The obtained crystals were filtered and washed withcold EtOAc and dried under reduced pressure to afford 2 (82 g, 58%) aswhite crystalline solid.

¹H-NMR: (400 MHz, CDCl₃): δ 7.35 (d, 5H), 7.20 (d, 1H), 7.00 (d, 1H),6.85 (d, 1H), 6.75 (d, 1H), 5.15-5.10 (m, 2H), 4.29 (d, 1H), 3.79 (d,3H), 3.59 (d, 1H), 3.57-3.49 (m, 2H), 3.10 (dd, 1H), 2.41-2.30 (m, 1H),2.09-2.00 (m, 2H), 1.70-1.65 (m, 1H), 1.37 (t, 1H).

LCMS (m/z): 381 [M⁺+1]

Synthesis of Benzyl 1-oxo-2,5-diazaspiro[3.4]octane-5-carboxylate (3)

A stirring solution of 2 (60 g, 0.16 mol) in MeCN (200 mL) and H₂O (30mL) was cooled to 0° C. and added a solution of CAN (86.5 g, 0.16 mol)in H₂O (30 mL). The reaction mixture was stirred at room temperature for1 h. The resulting mass was poured into ice cold water and the aqueouslayer was extracted with EtOAc (2×75 mL). The combined organic layerswere washed with saturated NaHCO₃ followed by brine, dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toget crude. The obtained material was purified by silica gel columnchromatography eluting with 70% EtOAc/n-hexane; finally obtainedmaterial was triturated with 10% EtOAc/n-hexane to afford 3 (15 g,36.5%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.99 (d, 1H), 7.42-7.30 (m, 5H), 5.19-5.00(m, 2H), 3.55 (d, 1H), 3.50-3.32 (m, 2H), 3.19 (dd, 1H), 2.18-2.00 (m,2H), 1.91-1.79 (m, 2H)

LCMS (m/z): 261 [M⁺+1]

Synthesis of Benzyl2-(2-ethoxy-2-oxoethyl)-1-oxo-2,5-diazaspiro[3.4]octane-5-carboxylate(4)

To a stirred solution of 3 (5 g, 19.23 mmol) in acetonitrile (100 mL)was added Cs₂CO₃ (8.1 g, 24.99 mmol) and ethyl 2-bromoacetate (3.2 mL,28.84 mmol) at RT and stirring was continued for 10 h at RT. Thevolatiles were evaporated under reduced pressure. The residue wasdiluted with water and extracted with EtOAc (2×50 mL). The separatedorganic layer was washed with brine, dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The obtained crude material waspurified by silica gel column chromatography eluting with 20%EtOAc/n-hexane to afford 4 (4.4 g, 66%) as syrup.

¹H-NMR: (500 MHz, CDCl₃): δ 7.38 (d, 5H), 5.19-5.04 (m, 2H), 4.49-3.17(m, 8H), 2.47-2.39 (m, 1H), 2.25-2.21 (m, 1H), 2.05-2.01 (m, 1H),1.95-1.92 (m, 1H), 1.30-1.25 (m, 3H).

LCMS (m/z): 345 [M⁺+1]

Synthesis of 2-(5-((Benzyloxy) carbonyl)-1-oxo-2, 5-diazaspiro [3.4]octan-2-yl) acetic acid (5)

To a stirred solution of 4 (0.2 g, 0.63 mmol) in THF: H₂O (6 mL, 5:1)was added LiOH.H₂O (66 mg, 1.57 mmol) at RT and stirred for 2 h. Aftercomplete consumption of the starting material (by TLC), the volatileswere evaporated under reduced pressure. The residue was diluted withwater, washed with ether, the aqueous layer was acidified to pH˜2 using2N HCl and extracted with EtOAc (2×50 mL). The organic layers werewashed with brine, dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to afford 5 (0.1 g).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.41 (d, 5H), 5.07-5.04 (m, 2H), 4.49-3.17(m, 8H), 2.21-2.09 (m, 2H), 1.95-1.92 (m, 2H).

LCMS (m/z): 319.4 [M⁺+1]

Synthesis of (2S,3R)-methyl 2-amino-3-hydroxybutanoate (Int-D)

To a stirring solution of (2S,3R)-2-amino-3-hydroxybutanoic acid (200 g,1.68 mol) in methanol (1.2 L) was added SOCl₂ (244 mL, 3.36 mol) dropwise at 0° C. and stirred for 1 h. The resulting reaction mixture wasrefluxed for 24 h. After consumption of the starting material (by TLC),the reaction mixture was warmed to RT and concentrated under reducedpressure and washed with n-hexane (2×50 mL). The residue was dissolvedin EtOH (1 L) and neutralized with Et₃N (471 mL, 3.36 mol) and againstirred for 2 h. The precipitated solid was filtered off, and theobtained filtrate was concentrated under reduced pressure to affordInt-D (195 g, 80%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.51 (br s, 3H), 4.13-4.10 (m, 1H), 3.91(br s, 1H), 1.20 (d, 3H).

LCMS (m/z): 134.1 [M⁺+1]

Synthesis of (2S, 3R)-2-amino-3-hydroxybutanamide (Int-E)

A solution of Int-D (190 g, 1.35 mol) in IPA (2 L) was taken inautoclave and purged NH₃ gas (7-8 kg) and stirred at 35° C. for 24 h.Then removed NH₃ gas and reaction mixture was concentrated under reducedpressure and added CH₂Cl₂ and filtered. Obtained solid was refluxed inEtOH for 1 h at 78° C. The reaction mass was filtered in heatingcondition and n-hexane was added to the filtrate and again stirred foranother 4 h. The precipitated solid was filtered and dried under reducedpressure to afford Int-E (160 g, 47%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.38 (br s, 1H), 7.02 (br s, 1H), 4.66 (brs, 1H), 3.77-3.70 (m, 1H), 2.93 (d, 1H), 2.72 (br m, 1H), 1.05 (d, 3H).

LCMS (m/z): 119.1 [M⁺+1]

UPLC (ELSD purity): 99.9%

Synthesis of Benzyl 2-(2-(((2S, 3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)amino)-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate (6)

To a stirring solution of 5 (1 g, 3.13 mmol) in CH₂Cl₂ (50 mL) was addedHATU (1.4 g, 3.70 mmol) followed by DIPEA (1.5 mL, 7.82 mmol) and Int-E(443 mg, 3.76 mmol) at 0° C. The reaction mixture was stirred at RT for16 h. After consumption of the starting material (by TLC), the reactionwas quenched with water and extracted with CH₂Cl₂ (2×100 mL). Theorganic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The crude was purified by column chromatography toafford 6 (0.6 g, 46%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.82-7.65 (m, 1H), 7.41-7.38 (m, 5H), 7.27(d, 1H), 7.12 (d, 1H), 5.10-5.01 (m, 2H), 4.89-4.85 (m, 1H), 4.25-3.99(m, 3H), 3.94-3.90 (m, 1H), 3.74-3.69 (m, 1H), 3.52-3.45 (m, 2H),2.22-2.09 (m, 2H), 1.92-1.79 (m, 2H), 1.27-1.25 (m, 1H), 1.09-1.01 (m,3H).

LCMS (m/z): 419.3 [M⁺+1]

Synthesis of (2S, 3R)-3-hydroxy-2-(2-(1-oxo-2,5-diazaspiro[3.4]octan-2-yl) acetamido) butanamide (7)

To a stirring solution of 6 (0.6 g, 1.43 mmol) in CH₃OH (20 mL) wasadded Pd/C (0.15 g) and stirred under H₂ atmosphere at RT for 6 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasfiltered through a pad of celite and washed with MeOH. The obtainedfiltrate was concentrated under reduced pressure to afford 7 (0.3 g,74%).

LCMS (m/z): 339.3 [M⁺+1]

Synthesis of 1-Methyl-1H-1,2,4-triazole-5-carboxylic acid (Int-C)

A solution of 1-methyl-1H-1,2,4-triazole SM 1 (1 g, 12.0 mmol) in dryTHF (10 mL) was cooled to −78° C. under N₂ atmosphere and added n-BuLi(7.5 mL, 12.0 mmol) slowly. The reaction mixture was stirred for 1 h,the reaction mixture was quenched with dry ice and stirred for 30 minand then diluted with water and EtOAc (10 mL). The separated organiclayer was concentrated under reduced pressure to afford Int-C (0.8 g,53%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.80 (s, 1H), 4.09 (s, 4H).

LCMS (m/z): 128.1 [M⁺+1]

Synthesis of(2S,3R)-3-hydroxy-2-(2-(5-(1-methyl-1H-1,2,4-triazole-5-carbonyl)-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamido) butanamide (Compound C)

To a stirring solution of 7 (0.25 g, 0.88 mmol) in CH₂Cl₂ (20 mL) wasadded HOBt (178 mg, 1.32 mmol), EDCI.HCl (0.2 g, 1.00 mmol) followed byDIPEA (0.4 mL, 2.20 mmol) and Int-C (134 mg, 1.05 mmol) at 0° C. Thereaction mixture was stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was concentrated underreduced pressure to obtain crude product. This material was purified bycolumn chromatography followed by prep-HPLC purification to affordCompound C (0.07 g, 21%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.09 (s, 1H), 7.89 (t, 1H), 7.25 (d, 1H),7.12 (t, 1H), 4.93 (s, 1H), 4.19-4.15 (m, 2H), 4.03 (s, 3H), 3.96-3.91(m, 4H), 3.44 (d, 1H), 2.25-2.20 (m, 3H), 1.97-1.91 (m, 2H), 1.07 (s,3H).

LCMS (m/z): 394.2 [M⁺+1]

HPLC Purity: 93%

Example 4—Synthesis of Compound D

Synthesis of 1, 3, 5-Tris (4-methoxybenzyl)-1, 3, 5-triazinane (1)

To a stirring solution of (4-methoxyphenyl) methanamine SM (200 g, 1.46mol) in EtOH (600 mL) at room temperature was added formaldehyde (33%aq, 105 mL) drop wise. The reaction mixture was stirred at roomtemperature for 1 h. After consumption of the starting material (byTLC), the reaction mixture was diluted with EtOAc (100 mL) and washedwith water (100 mL) followed by brine. The separated organic layer wasconcentrated under reduced pressure to obtain a crude product, which wasfinally washed with n-hexane to afford 1 (200 g, 30.6%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.18 (d, 6H), 6.84 (d, 6H), 3.73 (s, 9H),3.50 (s, 6H), 3.29 (s, 6H).

Synthesis of Benzyl 2-(chlorocarbonyl)pyrrolidine-1-carboxylate (Int-B)

To a stirring solution of Int-A (100 g, 0.40 mol) in CH₂Cl₂ (500 mL) wasadded catalytic amount of DMF (1 mL) and the reaction mixture was cooledto 0° C. To this oxalyl chloride (112.3 mL, 0.60 mol) was added dropwise and the reaction mixture was stirred at room temperature for 2 h.After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure to afford Int-B (100 g).This material was used directly in the next step without furtherpurification.

Synthesis of Benzyl 2-(4-methoxybenzyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (2)

To a stirring solution of Int-B (100 g, 0.37 mol) in dry CH₂Cl₂ (500 mL)was cooled to −40° C. and added Et₃N (210.2 mL, 1.50 mol) drop wise. Thereaction mixture was stirred at −40° C. for 45 min. A mixture of 1 (50g, 0.12 mol) in CH₂Cl₂ (150 mL) and BF₃.OEt₂ (47.6 g, 0.33 mol) wasadded drop wise at −40° C. The resulting reaction mixture was allowed tostir at RT for 16 h. After consumption of the starting material (asdetermined by TLC), the reaction mixture was washed with saturatedNaHCO₃ solution followed by brine. The separated organic layer was driedover anhydrous Na₂SO₄ and concentrated under reduced pressure. The crudematerial was dissolved in EtOAc and kept in the refrigerator forcrystallization. The obtained crystals were filtered and washed withcold EtOAc and dried under reduced pressure to afford 2 (82 g, 58%) aswhite crystalline solid.

¹H-NMR: (400 MHz, CDCl₃): δ 7.35 (d, 5H), 7.20 (d, 1H), 7.00 (d, 1H),6.85 (d, 1H), 6.75 (d, 1H), 5.15-5.10 (m, 2H), 4.29 (d, 1H), 3.79 (d,3H), 3.59 (d, 1H), 3.57-3.49 (m, 2H), 3.10 (dd, 1H), 2.41-2.30 (m, 1H),2.09-2.00 (m, 2H), 1.70-1.65 (m, 1H), 1.37 (t, 1H).

LCMS (m/z): 381 [M⁺+1]

Synthesis of Benzyl 1-oxo-2,5-diazaspiro[3.4]octane-5-carboxylate (3)

A stirring solution of 2 (60 g, 0.16 mol) in MeCN (200 mL) and H₂O (30mL) was cooled to 0° C. and added a solution of CAN (86.5 g, 0.16 mol)in H₂O (30 mL). The reaction mixture was stirred at room temperature for1 h. The resulting mass was poured into ice cold water and the aqueouslayer was extracted with EtOAc (2×75 mL). The combined organic layerswere washed with saturated NaHCO₃ followed by brine, dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toget crude. The obtained material was purified by silica gel columnchromatography eluting with 70% EtOAc/n-hexane. The purified materialwas triturated with 10% EtOAc/n-hexane to afford 3 (15 g, 36.5%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.99 (d, 1H), 7.42-7.30 (m, 5H), 5.19-5.00(m, 2H), 3.55 (d, 1H), 3.50-3.32 (m, 2H), 3.19 (dd, 1H), 2.18-2.00 (m,2H), 1.91-1.79 (m, 2H)

LCMS (m/z): 261 [M⁺+1]

Synthesis of Benzyl 2-(2-ethoxy-2-oxoethyl)-1-oxo-2,5-diazaspiro [3.4]octane-5-carboxylate (4)

To a stirred solution of 3 (5 g, 19.23 mmol) in acetonitrile (100 mL)was added Cs₂CO₃ (8.1 g, 24.99 mmol) and ethyl 2-bromoacetate (3.2 mL,28.84 mmol) at RT and stirring was continued for 10 h at RT. Thevolatiles were evaporated under reduced pressure. The residue wasdiluted with water and extracted with EtOAc (2×50 mL). The separatedorganic layer was washed with brine, dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The obtained crude material waspurified by silica gel column chromatography eluting with 20%EtOAc/n-hexane to afford 4 (4.4 g, 66%) in the form of a syrup.

¹H-NMR: (500 MHz, CDCl₃): δ 7.38 (d, 5H), 5.19-5.04 (m, 2H), 4.49-3.17(m, 8H), 2.47-2.39 (m, 1H), 2.25-2.21 (m, 1H), 2.05-2.01 (m, 1H),1.95-1.92 (m, 1H), 1.30-1.25 (m, 3H).

LCMS (m/z): 345 [M⁺+1]

Synthesis of 2-(5-((Benzyloxy) carbonyl)-1-oxo-2, 5-diazaspiro [3.4]octan-2-yl) acetic acid (5)

To a stirred solution of 4 (0.2 g, 0.63 mmol) in THF: H₂O (6 mL, 5:1)was added LiOH.H₂O (66 mg, 1.57 mmol) at RT and stirred for 2 h. Aftercomplete consumption of the starting material (by TLC), the volatileswere evaporated under reduced pressure. The residue was diluted withwater, washed with ether, the aqueous layer was acidified to pH˜2 using2N HCl and extracted with EtOAc (2×50 mL). The organic layers werewashed with brine, dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to afford 5 (0.1 g).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.41 (d, 5H), 5.07-5.04 (m, 2H), 4.49-3.17(m, 8H), 2.21-2.09 (m, 2H), 1.95-1.92 (m, 2H).

LCMS (m/z): 319.4 [M⁺+1]

Synthesis of (2S, 3R)-2-((tert-butoxycarbonyl) amino)-3-hydroxybutanoicacid (Int-D)

To a stirring solution of (2S, 3R)-2-amino-3-hydroxybutanoic acid (SM 2)(10 g, 83.9 mmol) in 1,4-dioxane/water (100 mL, 1:1)) was added NaHCO₃(21.1 g, 0.25 mol) followed by Boc-anhydride (21.9 mL, 0.101 mol) at 0°C. The reaction mixture was stirred at RT for 16 h. After consumption ofthe starting material (by TLC), the reaction mixture was diluted withwater and washed with EtOAc. The aqueous layer was acidified usingcitric acid solution (pH˜3-4) and then extracted with CH₂Cl₂ (2×150 mL).The separated organic extracts were dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to afford Int-D (15 g,crude). This material was directly used without further purification.

Synthesis of tert-butyl((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)carbamate (Int-E)

To a stirring solution of Int-D (5 g, 22.8 mmol) in CH₂Cl₂ (50 mL) wasadded EDCI.HCl (5.2 g, 27.3 mmol), HOBt (4.6 g, 34.2 mmol) followed byDIPEA (10.5 mL, 57 mmol) and pyrrolidine (1.945 g, 27.3 mmol) under N₂atmosphere at 0° C. The reaction mixture was stirred at RT for 16 h.After consumption of the starting material (by TLC), the reactionmixture was diluted with DCM and washed with water followed by saturatedNaHCO₃ and citric acid. The separated organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toobtain crude product. This material was purified by columnchromatography eluting with 2% MeOH/DCM to afford Int-E (3 g, 48%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 6.41 (d, 1H), 4.71 (d, 1H), 4.15 (t, 1H),3.94 (q, 1H), 3.63-3.42 (m, 2H), 3.24 (q, 1H), 1.90-1.81 (m, 4H), 1.38(s, 9H), 1.04 (s, 3H).

LCMS (m/z): 273.2 [M⁺+1]

Synthesis of (2S,3R)-2-amino-3-hydroxy-1-(pyrrolidin-1-yl)butan-1-one(Int-F)

To a stirred solution of Int-E (3 g, 11.0 mmol) in DCM (10 mL) was addedether-HCl (20 mL) at 0° C. under N₂ atmosphere. The reaction mixture wasstirred at RT for 4 h. The reaction mixture was concentrated underreduced pressure to get crude product, which was washed with ether toafford Int-F (2.0 g, 87%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.19 (br s, 3H), 3.98-3.91 (m, 2H),3.62-3.59 (m, 1H), 3.49-3.42 (m, 1H), 3.39-3.35 (m, 2H), 1.96-1.90 (m,4H), 1.17 (d, 3H).

LCMS (m/z): 173.3 [M⁺+1]

Synthesis of Benzyl 2-(2-(((2S, 3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl) amino)-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (6)

To a stirring solution of 5 (1 g, 3.14 mmol) in CH₂Cl₂ (50 mL) was addedEDCI (719 mg, 3.76 mmol), HOBt (635 mg, 4.71 mmol) followed by DIPEA(2.8 mL, 15.7 mmol) and C (784 mg, 3.77 mmol) and at 0° C. The reactionmixture was stirred at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction was quenched with water and extractedwith CH₂Cl₂ (2×50 mL). The organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure. The crude was purified bycolumn chromatography to afford 6 (0.8 g, 54%).

LCMS (m/z): 473.4 [M⁺+1]

Synthesis of N-((2S, 3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-2-(1-oxo-2, 5-diazaspiro[3,4] octan-2-yl)acetamide (7)

To a stirring solution of 6 (0.8 g, 1.69 mmol) in CH₃OH (60 mL) wasadded Pd/C (0.4 g) under N₂ atmosphere. The reaction mixture was stirredat RT for 4 h under H₂ atmosphere. After consumption of the startingmaterial (by TLC), the reaction mixture was filtered through a pad ofcelite and washed with MeOH. Obtained filtrate was concentrated underreduced pressure to afford 7 (0.5 g) as crude. This material wasdirectly used for the next step without further purification.

LCMS (m/z): 339.3 [M⁺+1]

Synthesis of 1-Methyl-1H-1,2,4-triazole-5-carboxylic acid (Int-C)

A solution of 1-methyl-1H-1,2,4-triazole SM 1 (1 g, 12.0 mmol) in dryTHF (10 mL) was cooled to −78° C. and n-BuLi (7.5 mL, 12.0 mmol) wasslowly added under N₂ atmosphere. The reaction mixture was stirred for 1h, quenched with dry ice and stirred for 30 min and then diluted withwater and EtOAc (10 mL). The separated organic layer was concentratedunder reduced pressure to afford Int-C (0.8 g, 53%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.80 (s, 1H), 4.09 (s, 3H).

LCMS (m/z): 128.1 [M⁺+1]

Synthesis of N-((2S, 3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-2-(5-(1-methyl-1H-1, 2, 4-triazole-5-carbonyl)-1-oxo-2,5-diazaspiro[3.4] octan-2-yl) acetamide (Compound D)

To a stirring solution of 7 (0.315 g, 2.48 mmol) in CH₂Cl₂ (60 mL) wasadded EDCI.HCl (336 mg, 1.76 mmol), HOBt (297 mg, 2.20 mmol), DIPEA(0.67 mL, 3.67 mmol) and Int-C (0.5 g, 1.47 mmol) at 0° C. The reactionmixture was stirred at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with DCM and washedwith water. The separated organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to obtain crudeproduct. This material was purified by column chromatography followed byprep-HPLC purification to afford Compound D (80 mg, 12%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.09 (s, 1H), 4.55-4.51 (m, 1H), 4.08 (d,2H), 3.97 (s, 2H), 3.87-3.84 (m, 3H), 3.70-3.55 (m, 2H), 3.45 (t, 1H),3.35-3.31 (m, 2H), 2.75 (s, 3H), 2.27-2.24 (m, 2H), 1.98-1.92 (m, 4H),1.85-1.84 (m, 2H), 1.19 (d, 3H).

LCMS (m/z): 448 [M⁺+1]

HPLC Purity: 94%

Example 5—Synthesis of Compound F

Synthesis of 1, 3, 5-Tris (4-methoxybenzyl)-1, 3, 5-triazinane (1)

To a stirring solution of SM (200 g, 1.46 mol) in EtOH (600 mL) at roomtemperature was added formaldehyde (33% aq, 105 mL) drop wise. Thereaction mixture was stirred at room temperature for 1 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with EtOAc (100 mL) and washed with water (100 mL) followed bybrine. The separated organic layer was concentrated under reducedpressure to obtained crude; which was triturated with n-hexane to afford1 (200 g, 30.6%) as white solid. ¹H-NMR: (500 MHz, DMSO-d₆): δ 7.18 (d,0.1=8.0 Hz, 6H), 6.81 (d, J=8.0 Hz, 6H), 3.71 (s, 9H), 3.50 (s, 6H),3.29 (s, 6H).

Synthesis of Benzyl 2-(4-methoxybenzyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (2)

To a stirring solution of 1 (45 g, 100 mmol) in CH₂Cl₂ (150 mL) wasadded BF₃.OEt₂ (37 mL, 301 mmol) drop wise at −40° C. To above stirringsolution Int-B (95 g, 342 mmol) in dry CH₂Cl₂ (500 mL) followed by Et₃N(210.2 mL, 1.50 mol) was added drop wise. The reaction mixture wasstirred at −40° C. for 45 min. The resulting reaction mixture wasallowed to stir at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was washed with saturated NaHCO₃solution (1×150 mL) followed by brine. The separated organic layer wasdried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Thecrude material was dissolved in EtOAc and kept in the refrigerator forcrystallization. Obtained crystals were filtered and washed with coldEtOAc (50 mL) and dried under vacuum to afford 2 (90 g, 65%) as whitecrystalline solid.

¹H-NMR: (500 MHz, DMSO-d6): 7.36-7.30 (m, 5H), 7.24 (d, J=8.0 Hz, 1H),7.06 (d, J=8.0 Hz, 1H), 6.90 (d, J=7.5 Hz, 1H), 6.81 (d, J=8.5 Hz, 1H),5.09 (s, 2H), 4.29 (s, 1H), 4.13, 3.96 (dd, J=15.5 Hz, 15.0 Hz, 1H),3.73 (s, 3H), 3.11 (t, J=5.0 Hz, 2H), 2.16-2.09 (m, 2H), 1.83-1.77 (m,2H), 1.20-1.15 (m, 2H)

LCMS m/z: 381 [M⁺+1]

Synthesis of Benzyl 1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate (3)

To a stirring solution of 2 (46 g, 121 mmol) in MeCN (460 mL) and H₂O(200 mL) were cooled to 0° C. and added a solution of CAN (199 g, 0.23mol) in H₂O (460 mL). The reaction mixture was stirred at roomtemperature for 1 h. The resulting mass was poured into ice cold water(100 mL) and the aqueous layer was extracted with EtOAc (2×200 mL). Thecombined organic layers were washed with saturated NaHCO₃ (1×150 mL)followed by brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford crude material waspurified by silica gel column chromatography eluting with EtOAc toobtain 3 (12 g, 38%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.90 (s, 1H), 7.36-7.29 (m, 5H), 5.10 (s,2H), 3.53 (d, J=4.5 Hz, 2H), 3.36-3.30 (m, 1H), 3.17, 3.13 (dd, J=5.0Hz, 5.0 Hz, 1H), 2.17-2.10 (m, 2H), 1.82-1.76 (m, 2H)

LCMS m/z: 261 [M⁺+1]

Synthesis of Benzyl 2-(2-ethoxy-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (4)

To a stirred solution of 3 (12 g, 46.1 mmol) in acetonitrile (120 mL)was added Cs₂CO₃ (37.6 g, 115.2 mmol) and ethyl 2-bromoacetate (7.7 mL,69.2 mmol) at RT and stirred for 16 h. After completion of reaction (byTLC), the volatiles were evaporated under reduced pressure. The residuewas diluted with water (50 mL) and extracted with EtOAc (2×100 mL). Theseparated organic layer was washed with brine, dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The obtained crudematerial was purified by silica gel column chromatography eluting with80% EtOAc/hexane to afford 4 (12.5 g, 78.6%) as pale brown syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.35-7.30 (m, 5H), 5.06 (s, 2H), 4.21 (s,1H), 4.18 (s, ¹H), 4.13-4.10 (m, 2H), 3.69 (d, J=4.5 Hz, 1H), 3.47-3.44(m, 3H), 2.16 (t, J=6.0 Hz, 2H), 1.87-1.80 (m, 2H), 1.21-1.14 (m, 3H)

LCMS m/z: 369.3 [M⁺+Na]

Synthesis of 2-(5-((benzyloxy) carbonyl)-1-oxo-2, 5-diazaspiro [3.4]octan-2-yl) acetic acid (5)

To a stirred solution of 4 (9.0 g, 26.0 mmol) in THF/H₂O (80 mL/30 mL)were added LiOH.H₂O (2.73 g, 65.0 mmol) at RT and stirred for 3 h. Afterconsumption of the starting material (by TLC), the volatiles wereevaporated under reduced pressure. The residue was diluted with water(25 mL), extracted with EtOAc (2×50 mL). The separated aqueous layer wasacidified to pH˜3 using 2N HCl and extracted with EtOAc (3×50 mL). Theorganic layers were dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to afford 5 (7.0 g, 85.3%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.5 (br s, 1H), 7.35-7.30 (m, 5H), 5.06(s, 2H), 4.21 (s, 1H), 4.18 (s, 1H), 3.69 (d, J=4.5 Hz, 1H), 3.47-3.44(m, 3H), 2.16 (t, J=6.0 Hz, 2H), 1.87-1.80 (m, 2H)

Synthesis of benzyl 2-(2-(((1S, 2R)-2-hydroxy-1-(1, 3, 4-oxadiazol-2-yl)propyl)amino)-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (6)

To a stirring solution of compound 5 (600 mg, 4.19 mmol) in DCM (20 mL)were added N, N-diisopropylethylamine (1.93 mL, 10.4 mmol), Int-I (1.60g, 5.02 mmol), followed by HATU (1.91 g, 5.02 mmol) at 0° C. and stirredat RT for 16 h. After consumption of the starting material (by TLC), thereaction mixture was diluted with water (20 mL). The separated organiclayer was washed with saturated NaHCO₃ solution (1×30 mL) followed bybrine solution (1×20 mL). The separated organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to afford 6(600 mg, 33.3%) as pale yellow syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.21 (s, 1H), 7.35-7.32 (m, 5H), 5.13 (d,J=4.5 Hz, 1H), 5.13 (s, 2H), 5.09-5.00 (m, 1H), 4.12-3.94 (m, 2H),3.70-3.33 (m, 6H), 2.16-2.11 (m, 4H), 1.08 (d, J=6.5 Hz, 3H)

LCMS m/z: 444.5 [M⁺+1]

Synthesis of N-((1S, 2R)-2-hydroxy-1-(1, 3, 4-oxadiazol-2-yl)propyl)-2-(1-oxo-2, 5-diazaspiro [3.4] octan-2-yl) acetamide (7)

To a stirring solution of 6 (600 mg, 1.35 mmol) in MeOH (10 mL) wereadded (50% wet) 10% Pd/C (200 mg) and stirred under H₂ atmosphere(balloon pressure) for 3 h at RT. After completion of reaction (by TLC),the reaction mixture was filtered through a pad of celite and trituratedwith EtOAc/MeOH (10 mL/10 mL). The filtrate was concentrated underreduced pressure to afford 7 (400 mg, crude) as yellow syrup.

¹H-NMR: (500 MHz, DMSO-d₅): δ 9.21 (s, 1H), 8.68 (s, 1H), 5.29-5.07 (m,1H), 4.10-3.91 (m, 2H), 3.61 (d, J=16.5 Hz, 1H), 3.40-3.31 (m, 1H),3.16-2.93 (m, 2H), 2.02-1.91 (m, 2H), 1.80-1.76 (m, 2H), 1.32-1.28 (m,1H), 1.24 (d, J=6.5 Hz, 1H), 1.09 (d, J=6.5 Hz, 3H)

LCMS m/z: 310.2 [M⁺+1];

Synthesis of N-((1S, 2R)-2-hydroxy-1-(1, 3, 4-oxadiazol-2-yl)propyl)-2-(5-(1-methyl-1H-1, 2, 4-triazole-5-carbonyl)-1-oxo-2,5-diazaspiro [3.4] octan-2-yl) acetamide (Compound F)

To a stirring solution of 7 (300 mg, 0.97 mmol) in DCM (30 mL) wereadded N, N-diisopropylethyl amine (0.44 mL, 2.42 mmol), Int-J (147 mg,1.16 mmol), followed by HATU (442 mg, 1.10 mmol) at 0° C. and stirred atRT for 16 h. After consumption of the starting material (by TLC), thereaction mixture was concentrated under reduced pressure to give crudeproduct, which was purified by column chromatography by 4% MeOH/DCM toafford yellow syrup which was further purified by preparative HPLC toafford Compound F (80 mg, 19.7%) as colorless thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.15 (s, 1H), 8.52 (t, J=8.0 Hz, 1H), 8.08(s, 1H), 5.19-5.16 (m, 1H), 4.04 (s, 2H), 4.02 (s, 3H), 3.94-3.84 (m,5H), 3.44-3.41 (m, 1H), 2.27-2.21 (m, 2H), 1.93-1.85 (m, 2H), 1.10-1.08(m, 3H)

LCMS m/z: 419 [M⁺+1]

HPLC: 96.64%

Synthesis of 1-methyl-1H-1, 2, 4-triazole-5-carboxylic acid (Int-J)

To a stirred solution of SM-1 (2.0 g, 24.0 mmol) in THF (20 mL) wasadded n-butyl lithium (19 mL, 12.0 mmol) at −78° C. dropwise and stirredfor 2 h. Then solid CO₂ (2 g) was added and stirred at −78° C. for 1 h.The reaction mass was stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was quenched with water(3 mL) and the obtained solid was filtered. The solid was trituratedwith diethylether/n-pentane (10 mL/10 mL). The white color solid wasdried under vacuum to afford Int-J (2.0 g, 65.7%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.70 (s, 1H), 4.01 (s, 3H)

LCMS m/z: 128.3 [M⁺+1]

Synthesis of (S)-1-((benzyloxy) carbonyl) pyrrolidine-2-carboxylic acid(Int-A)

To a stirring solution of L-proline (250 g, 2.17 mol) in water (1 L) wasadded Na₂CO₃ (576 g, 5.43 mol) and stirred for 1 h. After being cooledto 0° C., benzylchloroformate (50% in PhCH₃) (444 g, 2.61 mol) was addeddrop wise to the reaction mixture and again stirred for 1 h. Theresulting reaction mixture was warmed to RT and further stirred for 24h. After consumption of the starting material (by TLC), the reaction wasdiluted with water (1 L) and ether (1.5 L). The separated aqueous layerwas treated with PhCH₃ (1.5 L) and acidified with 6N HCl. The aqueouslayer was extracted with EtOAc (3×1.5 L), combined organic extracts werewashed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford Int-A (450 g, 84%) as paleyellow syrup.

¹H-NMR: (400 MHz, DMSO-d6): δ 12.71 (br s, 1H), 7.37-7.26 (m, 5H),5.07-4.99 (m, 2H), 4.25-4.15 (m, 1H), 3.45-3.34 (m, 2H), 2.25-2.14 (m,1H), 1.94-1.79 (m, 3H)

LCMS m/z: 250.4 [M⁺+1]

Synthesis of (S)-benzyl 2-(chlorocarbonyl) pyrrolidine-1-carboxylate(Int-B)

To a stirring solution of Int-A (2.5 g, 0.01 mol) in CH₂Cl₂ (50 mL) wasadded SOCl₂ (2.7 g, 0.02 mol) at 0° C. and stirred for 2 h. The reactionmixture was concentrated under reduced pressure to afford Int-B ascrude. This material was directly used for the next step without furtherpurification.

Synthesis of (2S, 3R)-methyl 2-amino-3-hydroxybutanoate (Int-C)

To a stirring solution of L-Thr-OH (60 g, 504 mmol) in CH₃OH (400 mL)was added thionyl chloride (70 mL, 972 mmol) at 0° C. and stirred at 75°C. for 6 h. After completion of starting material (by TLC), the reactionmixture was concentrated under reduced pressure to afford Int-C (60 g,crude). This material was directly used for the next step withoutfurther purification.

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.45 (s, 2H), 5.70 (s, 1H), 4.12-4.10 (m,1H), 3.90 (s, 1H), 3.73 (s, 3H), 1.20 (d, J=6.5 Hz, 3H).

Synthesis of (2S, 3R)-methyl 2-(((benzyloxy) carbonyl)amino)-3-hydroxybutanoate (Int-D)

To a stirring solution of NaHCO₃ (89 g, 1.065 mol) in water/1, 4 dioxane(150 mL/450 mL) were added Int-C (60 g, 355 mmol) at RT and stirred for30 min. The reaction mixture was cooled to 0° C. Cbz-Cl (60.7 mL, 426mmol) was added drop wise and stirred for 1 h. The reaction mixture wasstirred to RT and stirred for 16 h. After completion of startingmaterial (by TLC), the reaction mass was diluted with EtOAc (300 ml).The separated organic layer was washed with (2×200 mL) of saturatedNaHCO₃ solution followed by brine solution (2×100 mL). The organic layerwas dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to afford crude material was triturated with n-hexane anddiethylether (50 mL/50 mL) to afford Int-D (60 g, 63.8%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.37-7.30 (m, 5H), 7.20 (d, J=8.4 Hz, 1H),5.06 (s, 2H), 4.78 (d, J=6.8 Hz, 1H), 4.09-4.05 (m, 2H), 3.64 (s, 3H),1.09 (d, J=6.0 Hz, 3H)

LCMS 268.2 [M⁺+1]

Synthesis of (2S, 3R)-methyl 2-(((benzyloxy) carbonyl)amino)-3-((tert-butyldimethylsilyl) oxy) butanoate (Int-E)

To a stirring solution of Int-D (40 g, 149 mmol) in DMF (300 mL) wereadded DIPEA (69 mL, 374 mmol) TBDMS-Cl (30.91 mL, 179 mmol) at 0° C. andstirred at RT for 16 h. After completion of starting material (by TLC),the reaction mass was diluted with ether (200 ml). The separated organiclayer was washed with (2×200 mL) of saturated NaHCO₃ solution followedby brine solution (2×100 mL). The organic layer was dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure to afford crudematerial was purified by column chromatography eluting 10% EtOAc/hexaneto afford Int-E (40 g, 70.1%) as colorless syrup.

¹H-NMR: (400 MHz, CDCl₃): δ 7.39-7.32 (m, 5H), 5.43 (d, J=9.6 Hz, 1H),5.14 (s, 2H), 4.45-4.43 (m, 1H), 4.29-4.26 (m, 1H), 3.72 (s, 3H), 1.21(d, J=6.0 Hz, 3H), 0.83 (s, 9H), 0.09 (s, 6H)

LCMS m/z: 382.2[M⁺+1]

Synthesis of benzyl ((2S, 3R)-3-((tert-butyldimethylsilyl)oxy)-1-hydrazinyl-1-oxobutan-2-yl) carbamate

A solution of Int-E (20 g, 52.4 mmol) in EtOH (200 mL) was addedhydrazine hydrate (13.12 mL, 262 mmol), at RT and stirred at 90° C. for16 h. After completion of starting material (by TLC), ethanol wasevaporated under reduced pressure. The crude residue was diluted withwater (100 mL) and diethyl ether (200 mL). The separated organic layerwas dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure. Obtained crude material was purified by column chromatographyby eluting with 15% EtOAc/hexane to afford Int-F (4.0 g, 20%) ascolorless thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.10 (s, 1H), 7.36-7.30 (m, 5H), 6.83 (d,J=9.6 Hz, 1H), 5.02 (s, 2H), 4.19 (s, 2H), 4.05-4.02 (m, 1H), 3.97-3.93(m, 1H), 1.05 (d, 0.1=6.0 Hz, 3H), 0.81 (s, 9H), 0.01 (s, 6H)

Synthesis of benzyl ((1S, 2R)-2-((tert-butyldimethylsilyl) oxy)-1-(1, 3,4-oxadiazol-2-yl) propyl) carbamate (Int-G)

A solution of Int-F (4 g, 10.4 mmol) in triethyl orthoformate (40 mL)was added p-TSA (catalytic, 40 mg) at RT and after stirred at 120° C.for 3 h. After completion of starting material (by TLC),triethylorthoformate was evaporated under reduced pressure. The cruderesidue was purified by column chromatography eluting 10% EtOAc/hexaneto afford Int-G (2.8 g, 68%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.22 (s, 1H), 7.85 (d, J=9.5 Hz, 1H),7.36-7.31 (m, 5H), 5.05 (s, 2H), 4.96-4.93 (m, 1H), 4.25 (t, 0.1=6.0 Hz,1H), 1.23 (d, 0.1=6.0 Hz, 3H), 0.80 (s, 9H), 0.10 (s, 6H)

LCMS m/z: 392.4[M⁺+1]

Synthesis of (1S, 2R)-2-((tert-butyldimethylsilyl) oxy)-1-(1, 3,4-oxadiazol-2-yl) propan-1-amine (Int-H)

To a stirring solution of Int-G (2.8 g, 7.16 mmol) in methanol (30 mL)was added 50% wet 10% Pd/C (1.4 g) and stirred under H₂ atmosphere(balloon pressure) for 2 h at RT. The reaction mixture was filteredthrough a pad of celite and triturated with methanol (10 mL). Thefiltrate was concentrated under reduced pressure to afford Int-H (1.7 g,92%) as colorless syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.15 (s, 1H), 4.11 (t, J=5.0 Hz, 1H), 4.03(d, J=2.0 Hz, 1H), 2.05 (br s, 2H), 1.17 (d, J=6.0 Hz, 3H), 0.76 (s,9H), 0.02 (s, 6H)

LCMS m/z: 258.3 [M⁺+1]

Synthesis of (1S, 2R)-1-amino-1-(1, 3, 4-oxadiazol-2-yl) propan-2-ol(Int-I)

To a stirring solution of Int-H (500 mg, 1.94 mmol) in THF (6 mL) wasadded TBAF (1.01 mL) slowly at 0° C. and stirred at RT for 3 h. Aftercompletion of reaction (by TLC), the reaction mixture was evaporated anddiluted with EtOAc/H₂O (10 mL/2 mL). The separated organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to afford Int-I (120 mg, crude) as colorless thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.12 (s, 1H), 3.94 (d, J=4.5 Hz, 1H), 3.85(t, J=5.5 Hz, 1H), 3.17-3.13 (m, 3H), 1.05 (d, J=6.0 Hz, 3H).

Example 6—Synthesis of Compound G

Synthesis of 1, 3, 5-Tris (4-methoxybenzyl)-1, 3, 5-triazinane (1)

To a stirring solution of SM (200 g, 1.46 mol) in EtOH (600 mL) at roomtemperature was added formaldehyde (33% aq, 105 mL) drop wise. Thereaction mixture was stirred at room temperature for 1 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with EtOAc (100 mL) and washed with water (100 mL) followed bybrine. The separated organic layer was concentrated under reducedpressure to obtained crude; which was triturated with n-hexane to afford1 (200 g, 30.6%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.18 (d, J=8.0 Hz, 6H), 6.81 (d, J=8.0 Hz,6H), 3.71 (s, 9H), 3.50 (s, 6H), 3.29 (s, 6H).

Synthesis of Benzyl 2-(4-methoxybenzyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (2)

To a stirring solution of 1 (45 g, 100 mmol) in CH₂Cl₂ (150 mL) wasadded BF₃.OEt₂ (37 mL, 301 mmol) drop wise at −40° C. To above stirringsolution Int-B (95 g, 342 mmol) in dry CH₂Cl₂ (500 mL) followed by Et₃N(210.2 mL, 1.50 mol) was drop wise. The reaction mixture was stirred at−40° C. for 45 min. The resulting reaction mixture was allowed to warmto RT and stirred for 16 h. After consumption of the starting material(by TLC), the reaction mixture was washed with saturated NaHCO₃ solution(1×150 mL) followed by brine. The separated organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The crudematerial was dissolved in EtOAc and kept in the refrigerator forcrystallization. Obtained crystals were filtered and washed with coldEtOAc (50 mL) and dried under vacuum to afford 2 (90 g, 65%) as whitecrystalline solid.

¹H-NMR: (500 MHz, DMSO-d6): 7.36-7.30 (m, 5H), 7.24 (d, J=8.0 Hz, 1H),7.06 (d, J=8.0 Hz, 1H), 6.90 (d, J=7.5 Hz, 1H), 6.81 (d, J=8.5 Hz, 1H),5.09 (s, 2H), 4.29 (s, 1H), 4.13, 3.96 (dd, J=15.5 Hz, 15.0 Hz, 1H),3.73 (s, 3H), 3.11 (t, J=5.0 Hz, 2H), 2.16-2.09 (m, 2H), 1.83-1.77 (m,2H), 1.20-1.15 (m, 2H)

LCMS m/z: 381 [M⁺+1]

Synthesis of Benzyl 1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate (3)

To a stirring solution of 2 (46 g, 121 mmol) in MeCN (460 mL) and H₂O(200 mL) were cooled to 0° C. and added a solution of CAN (199 g, 0.23mol) in H₂O (460 mL). The reaction mixture was stirred at roomtemperature for 1 h. The resulting mass was poured into ice cold water(100 mL) and the aqueous layer was extracted with EtOAc (2×200 mL). Thecombined organic layers were washed with saturated NaHCO₁ (1×150 mL)followed by brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford crude material waspurified by silica gel column chromatography eluting with EtOAc toobtained 3 (12 g, 38%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.90 (s, 1H), 7.36-7.29 (m, 5H), 5.10 (s,2H), 3.53 (d, J=4.5 Hz, 2H), 3.36-3.30 (m, 1H), 3.17, 3.13 (dd, J=5.0Hz, 5.0 Hz, 1H), 2.17-2.10 (m, 2H), 1.82-1.76 (m, 2H)

LCMS m/z: 261 [M⁺+1]

Synthesis of Benzyl 2-(2-ethoxy-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (4)

To a stirred solution of 3 (12 g, 46.1 mmol) in acetonitrile (120 mL)was added Cs₂CO₁ (37.6 g, 115.2 mmol) and ethyl 2-bromoacetate (7.7 mL,69.2 mmol) at RT and stirred for 16 h at RT. After completion ofreaction (by TLC), the volatiles were evaporated under reduced pressure.The residue was diluted with water (50 mL) and extracted with EtOAc(2×100 mL). The separated organic layer was washed with brine, driedover anhydrous Na₂SO₄ and concentrated under reduced pressure. Theobtained crude material was purified by silica gel column chromatographyeluting with 80% EtOAc/hexane to afford 4 (12.5 g, 78.6%) as pale brownsyrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.35-7.30 (m, 5H), 5.06 (s, 2H), 4.21 (s,1H), 4.18 (s, 1H), 4.13-4.10 (m, 2H), 3.69 (d, J=4.5 Hz, 1H), 3.47-3.44(m, 3H), 2.16 (t, J=6.0 Hz, 2H), 1.87-1.80 (m, 2H), 1.21-1.14 (m, 3H)

LCMS m/z: 369.3 [M⁺+Na]

Synthesis of 2-(5-((benzyloxy) carbonyl)-1-oxo-2, 5-diazaspiro [3.4]octan-2-yl) acetic acid (5)

To a stirred solution of 4 (9.0 g, 26.0 mmol) in THF/H₂O (80 mL/30 mL)were added LiOH.H₂O (2.73 g, 65.0 mmol) at RT and stirred for 3 h. Afterconsumption of the starting material (by TLC), the volatiles wereevaporated under reduced pressure. The residue was diluted with water(25 mL), extracted with EtOAc (2×50 mL). The separated aqueous layer wasacidified to pH˜3 using 2N HCl and extracted with EtOAc (3×50 mL). Theorganic layers were dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to afford 5 (7.0 g, 85.3%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.5 (br s, 1H), 7.35-7.30 (m, 5H), 5.06(s, 2H), 4.21 (s, 1H), 4.18 (s, 1H), 3.69 (d, J=4.5 Hz, 1H), 3.47-3.44(m, 3H), 2.16 (t, J=6.0 Hz, 2H), 1.87-1.80 (m, 2H)

Synthesis of benzyl 2-(2-((2-((tert-butyldimethylsilyl)oxy)-1-(Pyrimidin-2-yl) propyl)amino)-2-oxoethyl)-1-oxo-2, 5-diazaspiro[3.4] octane-5-carboxylate (6)

To a stirring solution of 5 (1.3 g, 4.08 mmol) in DCM (30 mL) were addedN, N-diisopropylethylamine (2.1 mL, 12.2 mmol), Int J (1.09 g, 4.08mmol), followed by HOBt (938 mg, 6.13 mmol), EDCI.HCl (1.1 g, 6.13 mmol)at 0° C. and stirred at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with water (30 mL).The separated organic layer was washed with brine solution (1×50 mL).The separated organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to afford crude which was purifiedby column chromatography by eluting 4% MeOH/DCM to obtained 6 (1.5 g,65%) as yellow thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.80-8.74 (m, 2H), 7.39-7.34 (m, 6H), 5.09(s, 2H), 4.93 (t, J=4.8 Hz, 1H), 4.38-4.19 (m, 1H), 4.05-3.68 (m, 2H),3.49-3.39 (m, 4H), 2.20-2.11 (m, 2H), 1.86-1.85 (m, 2H), 1.19-1.10 (m,4H), 0.65 (s, 9H), −0.07 (s, 3H), −0.03 (s, 3H)

LCMS m/z: 568 [M⁺+1]

Synthesis of N-(2-((tert-butyldimethylsilyl) oxy)-1-(pyrimidin-2-yl)propyl)-2-(1-oxo-2, 5-diazaspiro [3.4] octan-2-yl) acetamide (7)

To a stirring solution of 6 (500 mg, 0.88 mmol) in EtOAc (25 mL) wasadded (50% wet) 10% Pd/C (250 mg) and stirred under H₂ atmosphere(balloon pressure) at RT for 7 h. After completion of reaction (by TLC),the reaction mixture was filtered through a pad of celite. The filtratewas concentrated under reduced pressure to afford 7 (320 mg, crude) asyellow thick syrup. This compound was used directly for next stepwithout any purification.

LCMS m/z: 434.5 [M⁺+1]

Synthesis of N-(2-((tert-butyldimethylsilyl) oxy)-1-(pyrimidin-2-yl)propyl)-2-(5-(1-methyl-1H-1, 2, 4-triazole-5-carbonyl)-1-oxo-2,5-diazaspiro [3.4] octan-2-yl) acetamide (8)

To a stirring solution of Int-C (93 mg, 0.73 mmol) in DCM (20 mL) wereadded N, N-diisopropylethylamine (0.4 mL, 2.21 mmol), compound 7 (320mg, 0.73 mmol), followed by EDCI.HCl (211 mg, 1.10 mmol), HOBt (170 mg,1.10 mmol) at 0° C. and stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was evaporated underreduced pressure and the obtained crude was purified by columnchromatography by eluting 4% MeOH/DCM to afford 8 (210 mg, crude) asyellow thick syrup. This compound was used directly for next stepwithout any purification.

LCMS m/z: 543.5 [M⁺+1]

Synthesis of N-(2-hydroxy-1-(pyrimidin-2-yl)propyl)-2-(5-(1-methyl-1H-1, 2, 4-triazole-5-carbonyl)-1-oxo-2,5-diazaspiro [3.4] octan-2-yl) acetamide (Compound G)

To a stirring solution of compound 8 (210 mg, 0.38 mmol) in THF (5 mL)was added TBAF in THF (0.77 ml, 0.76 mmol) at 0° C. and stirred at RTfor 3 h. After consumption of the starting material (by TLC), thereaction mixture was evaporated under reduced pressure and the obtainedcrude was purified by column chromatography by eluting 4% MeOH/DCMfollowed by preparative TLC to afford Compound G (70 mg, 42%) as yellowthick syrup. ¹H-NMR: (400 MHz, CD₃OD): δ 8.75-8.70 (m, 2H), 7.95 (s,1H), 7.36-7.30 (m, 1H), 5.16-5.13 (m, 1H), 4.53 (s, 1H), 4.41-4.43 (m,2H), 4.29-4.03 (m, 1H), 3.99 (s, 3H), 3.96-3.81 (m, 2H), 3.58-3.54 (m,1H), 2.37-2.34 (m, 2H), 2.09-2.00 (m, 2H), 1.22 (d, J=6.4 Hz, 3H)

LCMS m/z: 429 [M⁺+1]

Synthesis of (S)-1-((benzyloxy) carbonyl) pyrrolidine-2-carboxylic acid(Int-A)

To a stirring solution of L-proline (250 g, 2.17 mol) in water (1 L) wasadded Na₂CO₃ (576 g, 5.43 mol) and stirred for 1 h. After being cooledto 0° C., benzylchloroformate (50% in PhCH₃) (444 g, 2.61 mol) was addeddrop wise to the reaction mixture and again stirred for 1 h. Theresulting reaction mixture was warmed to RT and further stirred for 24h. After consumption of the starting material (by TLC), the reaction wasdiluted with water (1 L) and ether (1.5 L). The separated aqueous layerwas treated with PhCH₃ (1.5 L) and acidified with 6N HCl. The aqueouslayer was extracted with EtOAc (3×1.5 L), combined organic extracts werewashed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford Int-A (450 g, 84%) as paleyellow syrup. ¹H-NMR: (400 MHz, DMSO-d₆): δ 12.71 (br s, 1H), 7.37-7.26(m, 5H), 5.07-4.99 (m, 2H), 4.25-4.15 (m, 1H), 3.45-3.34 (m, 2H),2.25-2.14 (m, 1H), 1.94-1.79 (m, 3H).

LCMS m/z: 250.4 [M⁺+1]

Synthesis of (S)-benzyl 2-(chlorocarbonyl) pyrrolidine-1-carboxylate(Int-B)

To a stirring solution of Int-A (2.5 g, 0.01 mol) in CH₂Cl₂ (50 mL) wasadded SOCl₂ (2.7 g, 0.02 mol) at 0° C. and stirred for 2 h. The reactionmixture was concentrated under reduced pressure to afford Int-B ascrude. This material was directly used for the next step without furtherpurification.

Synthesis of 1-(pyrimidin-2-yl) propan-1-one (Int-D)

To a stirring solution of SM-2 (20 g, 190 mmol) in THF (200 mL) wasadded ethyl magnesium bromide (1M in THF, 227 mL, 228 mmol) at 0° C. for1 h. After completion of starting material (by TLC), the reactionmixture was diluted with saturated ammonium chloride solution and EtOAc(150 mL). The separated organic layer was washed with brine solution(2×100 mL). The extracted organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to afford crudematerial which was purified by column chromatography eluting 20%EtOAc/hexane to afford Int-D (15 g, 57%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.00 (d, J=5.2 Hz, 2H), 7.70 (t, J=4.8 Hz,1H), 3.20-3.15 (m, 2H), 1.09 (t, J=7.2 Hz, 3H)

LCMS m/z: 137 [M⁺+1]

Synthesis of (Z)-2-(1-((triethylsilyl) oxy) prop-1-en-1-yl) pyrimidine(Int-E)

To a stirring solution of Int-D (15 g, 110 mmol) in THF (100 mL) wasadded LiHMDS (1M in THF, 220 mL, 220 mmol) slowly at 0° C. and stirredfor 30 min. After added chloro triethylsilane (24.8 g, 165 mmol) in THF(50 mL) dropwise at 0° C. and stirred 1 h. After completion of startingmaterial (by TLC), the reaction mixture was diluted with saturatedammonium chloride solution (50 mL) and EtOAc (150 mL). The separatedorganic layer was extracted with brine solution (2×100 mL). Theseparated organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford crude material which waspurified by column chromatography eluting 5% EtOAc/hexane to affordInt-E (20 g, 74%) as yellow thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.75 (d, J=4.8 Hz, 2H), 7.32 (t, J=4.8 Hz,1H), 6.36-6.31 (m, 1H), 1.77 (d, J=7.2 Hz, 3H), 0.95-0.87 (m, 9H),0.71-0.65 (m, 6H)

Synthesis of 2-bromo-1-(pyrimidin-2-yl) propan-1-one (Int-F)

To a stirring solution of Int-E (20 g, 80 mmol) in THF/H₂O (160 mL/40mL) were added N-bromosuccinimide (10.2 g, 88 mmol) slowly at RT andstirred for 2 h. After completion of starting material (by TLC), thereaction mixture was diluted with H₂O and EtOAc (100 ml/150 mL). Theseparated organic layer was washed with brine solution (2×100 mL), driedover anhydrous Na₂SO₄, filtered and concentrated under reduced pressureto afford crude material which was purified by column chromatographyeluting 30% EtOAc/hexane to afford Int-F (15 g, 87%) as yellow thicksyrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.06 (d, J=4.8 Hz, 2H), 7.75 (t, J=4.8 Hz,1H), 5.97-5.92 (m, 1H), 1.83 (d, J=6.4 Hz, 3H)

Synthesis of 2-hydroxy-1-(pyrimidin-2-yl) propan-1-one (Int-G)

To a stirring solution of Int-F (15 g, 69 mmol) in MeOH (240 mL) wasadded sodium formate (18.9 g, 279 mmol) and stirred the reaction mass at70° C. for 8 h. After completion of reaction (by TLC), the reactionmixture was evaporated under reduced pressure to give crude product,which was purified by column chromatography eluting 5% MeOH/DCM toafford Int-G (5.5 g, 51%) as colorless liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.73 (d, J=5.2 Hz, 2H), 7.55 (t, J=4.8 Hz,1H), 5.28-5.26 (m, 1H), 1.24 (d, J=6.4 Hz, 1H), 0.99 (d, J=6.4 Hz, 3H).

Synthesis of (2-((tert-butyldimethylsilyl) oxy)-1-(pyrimidin-2-yl)propan-1-one (Int-H)

To a stirring solution of Int-G (5.5 g, 36 mmol) in DCM (150 mL) wereadded imidazole (4.9 g, 72 mmol), DMAP (880 mg, 0.72 mmol) at 0° C. andstirred for 10 min. After added TBDMS-Cl (8.1 g, 54 mmol) at 0° C. andstirred at RT for 6 h. After completion of starting material (by TLC),diluted the reaction mass with H₂O (50 ml). The separated organic layerwas washed with brine solution (2×50 mL). The organic layer was driedover anhydrous Na₂SO₄, filtered and concentrated under reduced pressureto afford crude material which was purified by column chromatographyeluting 30% EtOAc/hexane to afford Int-H (3 g, 31%) as an off-whitesolid.

¹H-NMR: (400 MHz, CDCl₃): δ 9.00 (d, J=5.2 Hz, 2H), 7.71 (t, J=4.8 Hz,1H), 5.47-5.42 (m, 1H), 1.35 (d, J=6.8 Hz, 3H), 0.79 (s, 9H), 0.05 (s,6H).

Synthesis of (2-((tert-butyldimethylsilyl) oxy)-1-(pyrimidin-2-yl)propan-1-amine (Int-I)

To a stirring solution of Int-H (3 g, 11.2 mmol) in MeOH (50 mL) wereadded sodium acetate (1.8 g, 22.5 mmol), ammonium carbonate (8.8 g, 56.3mmol), AcOH (0.6 mL, 11.2 mmol) at RT and stirred at 70° C. for 2 h. Thereaction mixture was cooled to RT and sodium cyanoborohydride (1.39 g,22.5 mmol) was added and stirred at 70° C. for 6 h. After completion ofstarting material (by TLC), MeOH was evaporated and the crude residuewas diluted with DCM/H₂O (50 ml/50 mL). The separated organic layer waswashed with brine (2×50 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford crude material waspurified by column chromatography eluting 5% MeOH/DCM to afford Int-I(2.4 g, 80%) as semi solid.

¹H-NMR: (400 MHz, CDCl₃): δ 8.83 (d, J=4.8 Hz, 2H), 7.40 (t, J=5.2 Hz,1H), 4.13 (t, J=6.4 Hz, 2H), 3.90 (d, J=6.4 Hz, 2H), 1.12 (d, J=6.4 Hz,3H), 0.70 (s, 9H), 0.02 (s, 6H).

Synthesis of 1-methyl-1H-1, 2, 4-triazole-5-carboxylic acid (C)

To a stirred solution of SM-1 (2.0 g, 24.0 mmol) in THF (20 mL) wasadded n-butyl lithium (19 mL, 12.0 mmol) at −78° C. dropwise and stirredfor 2 h and then quenched by addition of solid CO₂ (2 g) at −78° C. Thereaction mixture was allowed to warm to room temperature and stirred for16 h. After consumption of the starting material (by TLC), the reactionmixture was treated with water (3 mL) and the obtained solid wasfiltered. The solid was triturated with diethylether/n-pentane (10 mL/10mL), dried under vacuum to afford Int-C (2.0 g, 65.7%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.70 (s, 1H), 4.01 (s, 3H).

LCMS m/z: 128.3 [M⁺+1]

Example 7—Synthesis of Compound H

Synthesis of 1,3,5-Tris (4-methoxybenzyl)-1, 3, 5-triazinane (1)

To a stirring solution of (4-methoxyphenyl) methanamine SM (200 g, 1.46mol) in EtOH (600 mL) at room temperature was added formaldehyde (33%aq, 105 mL) drop wise. The reaction mixture was stirred at roomtemperature for 1 h. After consumption of the starting material (byTLC), the reaction mixture was diluted with EtOAc (100 mL) and washedwith water (100 mL) followed by brine. The separated organic layer wasconcentrated under reduced pressure to obtain crude; which was washedwith n-hexane to afford 1 (200 g, 30.6%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.18 (d, 6H), 6.84 (d, 6H), 3.73 (s, 9H),3.50 (s, 6H), 3.29 (s, 6H).

Synthesis of Benzyl 2-(4-methoxybenzyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (2)

To a stirring solution of Int-D (100 g, 0.37 mol) in dry CH₂Cl₂ (500 mL)cooled to −40° C. was added Et₃N (210.2 mL, 1.50 mol) drop wise. Thereaction mixture was stirred at −40° C. for 45 min. To this, a mixtureof compound 1 (50 g, 0.12 mol) in CH₂Cl₂ (150 mL) and BF₃.OEt₂ (47.6 g,0.33 mol) was added drop wise at −40° C. The resulting reaction mixturewas allowed to stir at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was washed with saturated NaHCO₃solution followed by brine. The separated organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The crudematerial was dissolved in EtOAc and kept in the refrigerator forcrystallization. Obtained crystals were filtered and washed with coldEtOAc and dried under vacuum to afford 2 (82 g, 58%) as whitecrystalline solid.

¹H-NMR: (400 MHz, CDCl₃): δ 7.35 (d, 5H), 7.20 (d, 1H), 7.00 (d, 1H),6.85 (d, 1H), 6.75 (d, 1H), 5.15-5.10 (m, 2H), 4.29 (d, 1H), 3.79 (d,3H), 3.59 (d, 1H), 3.57-3.49 (m, 2H), 3.10 (dd, 1H), 2.41-2.30 (m, 1H),2.09-2.00 (m, 2H), 1.70-1.65 (m, 1H), 1.37 (t, 1H)

LCMS m/z: 381 [M⁺+1]

Synthesis of Benzyl 1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate (3)

A stirring solution of 2 (60 g, 0.16 mol) in MeCN (200 mL) and H₂O (30mL) was cooled to 0° C. and a solution of CAN (86.5 g, 0.48 mol) in H₂O(30 mL) was added. The reaction mixture was stirred at room temperaturefor 1 h. The resulting mass was poured into ice cold water and theaqueous layer was extracted with EtOAc (2×75 mL). The combined organiclayers were washed with saturated NaHCO₃ followed by brine, dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toget crude. Obtained material was purified by silica gel columnchromatography eluting with 70% EtOAc/hexane; obtained material wastriturated with 10% EtOAc/hexane to afford 3 (15 g, 36.5%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.99 (d, 1H), 7.42-7.30 (m, 5H), 5.19-5.00(m, 2H), 3.55 (d, 1H), 3.50-3.32 (m, 2H), 3.19 (dd, 1H), 2.18-2.00 (m,2H), 1.91-1.79 (m, 2H)

LCMS m/z: 261 [M⁺+1]

Synthesis of Benzyl 2-(2-ethoxy-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (4)

To a stirred solution of 3 (5 g, 19.23 mmol) in acetonitrile (100 mL)was added Cs₂CO₃ (15.6 g, 48 mmol) and ethyl 2-bromoacetate (3.2 mL,28.84 mmol) at RT and stirring was continued at RT for 10 h. Thevolatiles were evaporated under reduced pressure. The residue wasdiluted with water and extracted with EtOAc (2×50 mL). The separatedorganic layer was washed with brine, dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The obtained crude material waspurified by silica gel column chromatography eluting with 30%EtOAc/hexane to afford 4 (3.7 g, 56%) as pale yellow syrup.

¹H-NMR: (500 MHz, CDCl₃): δ 7.38 (d, 5H, 5.19-5.04 (m, 2H), 4.49-3.17(m, 8H), 2.47-2.39 (m, 1H), 2.25-2.21 (m, 1H), 2.05-2.01 (m, 1H),1.95-1.92 (m, 1H), 1.30-1.25 (m, 3H)

LCMS (m/z): 345 [M⁺+1].

Synthesis of 2-(5-((benzyloxy)carbonyl)-1-oxo-2,5-diazaspiro [3.4]octan-2-yl)acetic acid (5)

To a stirred solution of 4 (2 g, 5.783 mmol) in THF: H₂O (24 mL, 5:1)was added LiOH.H₂O (606 mg, 14.4 mmol) at RT and stirred for 411. Aftercomplete consumption of the starting material (by TLC), the volatileswere evaporated under reduced pressure. The residue was diluted withwater, washed with ether, the aqueous layer was acidified to pH˜3-4using citric acid and extracted with EtOAc (2×50 mL). The organic layerswere washed with brine, dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to afford 5 (1.5 g, 83%) as pale yellow syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.41 (d, 5H), 5.07-5.04 (m, 2H), 4.49-3.17(m, 8H), 2.21-2.09 (m, 2H), 1.95-1.92 (m, 2H)

LCMS (m/z): 319.4 [M⁺+1]

Synthesis of Benzyl 2-(2-(((2S, 3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)amino)-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate (6)

To a stirring solution of 5 (1 g, 3.13 mmol) in CH₂Cl₂ (50 mL) was addedHATU (1.4 g, 3.70 mmol) followed by DIPEA (1.5 mL, 7.82 mmol) and Int-B(443 mg, 3.76 mmol) at 0° C. The reaction mixture was stirred at RT for16 h. After consumption of the starting material (by TLC), the reactionwas quenched with water and extracted with CH₂Cl₂ (2×100 mL). Theorganic layer was dried over anhydrous Na₂SO₄ and concentrated undervacuum. The crude was purified by column chromatography to afford 6 (0.6g, 46%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.82-7.65 (m, 1H), 7.41-7.38 (m, 5H), 7.27(d, 1H), 7.12 (d, 1H), 5.10-5.01 (m, 2H), 4.89-4.85 (m, 1H), 4.25-3.99(m, 3H), 3.94-3.90 (m, 1H), 3.74-3.69 (m, 1H), 3.52-3.45 (m, 2H),2.22-2.09 (m, 2H), 1.92-1.79 (m, 2H), 1.27-1.25 (m, 1H), 1.09-1.01 (m,3H)

LCMS m/z: 419.3 [M⁺+1]

Synthesis of(2S,3R)-2-(2-(5-acetyl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamido)-3-hydroxybutanamide (Compound H)

To a stirring solution of 6 (1 g, 2.39 mmol) in EtOAc (50 mL) was addedacetic anhydride (0.48 g, 4.78 mmol) followed by Pd/C (0.5 g) under N₂atmosphere. The reaction mixture was stirred at RT for 16 h under H₂atmosphere. After consumption of the starting material (by TLC), thereaction mixture was filtered through a pad of celite and washed withEtOAc (20 mL). Obtained filtrate was concentrated under reduced pressureto afford crude compound was purified by column chromatography. Theobtained mixture of compound was purified by chiral preparative HPLC toafford Compound H fraction-I (0.075 g), fraction-II (0.062 g) asoff-white solids.

¹H-NMR (Fr-I): (400 MHz, DMSO-d₆): δ 7.87 (d, J=8.0 Hz, 1H), 7.24 (s,1H), 7.02 (s, 1H), 4.84 (d, J=7.2 Hz, 1H), 4.10-4.03 (m, 2H), 3.96 (s,2H), 3.79 (d, J=5.2 Hz, 1H), 3.61-3.56 (m, 1H), 3.51-3.45 (m, 2H),2.19-2.15 (m, 2H), 2.00 (s, 3H), 1.92-1.87 (m, 2H), 1.05 (d, J=6.4 Hz,3H)

LCMS m/z: 327.3 [M⁺+1]

HPLC Purity Fr-I (91.20%),

¹H-NMR (Fr-II): (400 MHz, DMSO-d₆): δ 7.85 (d, J=8.4 Hz, 1H), 7.17 (s,1H), 7.07 (s, 1H), 4.79 (d, J=6.8 Hz, 1H), 4.09-4.06 (m, 2H), 3.97 (s,2H), 3.70 (d, J=4.8 Hz, 1H), 3.60-3.55 (m, 1H), 3.51-3.45 (m, 2H),2.18-2.13 (m, 2H), 1.99 (s, 3H), 1.92-1.85 (m, 2H), 1.05 (d, J=6.4 Hz,3H)

LCMS m/z: 327.4 [M⁺+1]

HPLC Purity Fr-II (97.03%)

Synthesis of (2S,3R)-methyl 2-amino-3-hydroxybutanoate (Int-A)

To a stirring solution of (2S, 3R)-2-amino-3-hydroxybutanoic acid (200g, 1.68 mol) in methanol (1.2 L) was added SOCl₂ (244 mL, 3.36 mol) dropwise at 0° C. and stirred for 1 h. The resulting reaction mixture wasrefluxed for 24 h. After consumption of the starting material (by TLC),the reaction mixture was warmed to RT and concentrated under vacuum anddecanted with n-hexane (2×50 mL). The residue was dissolved in EtOH (1L) and neutralized with Et₃N (471 mL, 3.36 mol) and again stirred for 2h. The precipitated solid was filtered off; obtained filtrate wasconcentrated under vacuum to afford Int-A (195 g, 80%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.51 (br s, 3H), 4.13-4.10 (m, 1H), 3.91(br s, 1H), 1.20 (d, 3H)

LCMS m/z: 134.1 [M⁺+1]

Synthesis of (2S, 3R)-2-amino-3-hydroxybutanamide (Int-B)

A solution of Int-A (190 g, 1.35 mol) in IPA (2 L) was taken inautoclave and purged NH₃ gas (7-8 kg) and stirred at 35° C. for 24 h.After completion of the reaction, NH₃ was expelled and reaction mixturewas concentrated under reduced pressure and added CH₂Cl₂ and filtered.Obtained solid was refluxed in EtOH for 1 h at 78° C. The reaction masswas filtered in heating condition and n-hexane was added to the filtrateand again stirred for another 4 h. Obtained precipitated solid wasfiltered and dried under vacuum to afford Int-B (160 g, 47%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.38 (br s, 1H), 7.02 (br s, 1H), 4.66 (brs, 1H), 3.77-3.70 (m, 1H), 2.93 (d, 1H), 2.72 (br m, 1H), 1.05 (d, 3H)

LCMS m/z: 119.1 [M⁺+1]

UPLC (ELSD purity): 99.9%

Synthesis of (S)-1-((benzyloxy) carbonyl) pyrrolidine-2-carboxylic acid(Int-C)

To a stirring solution of L-proline (250 g, 2.17 mol) in water (1 L) wasadded Na₂CO₃ (576 g, 5.43 mol) and stirred for 1 h. After being cooledto 0° C., benzylchloroformate (50% in PhCH₃) (444 g, 2.61 mol) was addeddrop wise to the reaction mixture and again stirred for 1 h. Theresulting reaction mixture was warmed to RT and further stirred for 24h. After consumption of the starting material (by TLC), the reaction wasdiluted with water (1 L) and ether (1.5 L). The separated aqueous layerwas treated with PhCH₃ (1.5 L) and acidified using 6N HCl. The aqueouslayer was extracted with EtOAc (3×1.5 L), combined organic extracts werewashed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford Int-C (450 g, 84%) aslight yellow syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.71 (br s, 1H), 7.37-7.26 (m, 5H),5.07-4.99 (m, 2H), 4.25-4.15 (m, 1H), 3.45-3.34 (m, 2H), 2.25-2.14 (m,1H), 1.94-1.79 (m, 3H)

LCMS m/z: 250.4 [M⁺+1]

Benzyl 2-(chlorocarbonyl)pyrrolidine-1-carboxylate (Int-D)

To a stirring solution of 1-((benzyloxy) carbonyl)pyrrolidine-2-carboxylic acid (Int-C) (100 g, 0.40 mol) in CH₂Cl₂ (500mL) was added catalytic amount of DMF (1 mL) and the reaction mixturewas cooled to 0° C. To this oxalyl chloride (112.3 mL, 0.60 mol) wasadded drop wise and the reaction mixture was stirred at room temperaturefor 2 h. After consumption of the starting material (by TLC), thereaction mixture was concentrated under reduced pressure to afford Int-D(100 g) as crude. This material was directly used for the next stepwithout further purification.

Example 8—Synthesis of Compound I

Synthesis of 1, 3, 5-Tris (4-methoxybenzyl)-1, 3, 5-triazinane (1)

To a stirring solution of (4-methoxyphenyl) methanamine SM (200 g, 1.46mol) in EtOH (600 mL) at room temperature was added formaldehyde (33%aq, 105 mL) drop wise. The reaction mixture was stirred at roomtemperature for 1 h. After consumption of the starting material (byTLC), the reaction mixture was diluted with EtOAc (100 mL) and washedwith water (100 mL) followed by brine. The separated organic layer wasconcentrated under reduced pressure to obtain crude; which was finallywashed with n-hexane to afford 1 (200 g, 30.6%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.18 (d, 6H), 6.84 (d, 6H), 3.73 (s, 9H),3.50 (s, 6H), 3.29 (s, 6H).

Synthesis of Benzyl 2-(4-methoxybenzyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (2)

To a stirring solution of Int-B (100 g, 0.37 mol) in dry CH₂Cl₂ (500 mL)was cooled to −40° C. and Et₃N (210.2 mL, 1.50 mol) was added drop wise.The reaction mixture was stirred at −40° C. for 45 min. To this, amixture of 1 (50 g, 0.12 mol) and BF₃OEt₂ (47.6 g, 0.33 mol) in CH₂Cl₂(150 mL) was added drop wise at −40° C. The resulting reaction mixturewas allowed to stir at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was washed with saturated NaHCO₃solution followed by brine. The separated organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The crudematerial was dissolved in EtOAc and kept in the refrigerator forcrystallization. Obtained crystals were filtered and washed with coldEtOAc and dried under vacuum to afford 2 (82 g, 58%) as whitecrystalline solid.

¹H-NMR: (400 MHz, CDCl₃): δ 7.35 (d, 5H), 7.20 (d, 1H), 7.00 (d, 1H),6.85 (d, 1H), 6.75 (d, 1H), 5.15-5.10 (m, 2H), 4.29 (d, 1H), 3.79 (d,3H), 3.59 (d, 1H), 3.57-3.49 (m, 2H), 3.10 (dd, 1H), 2.41-2.30 (m, 1H),2.09-2.00 (m, 2H), 1.70-1.65 (m, 1H), 1.37 (t, 1H)

LCMS m/z: 381 [M⁺+1]

Synthesis of Benzyl 1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate (3)

To a stirring solution of 2 (60 g, 0.16 mol) in MeCN (200 mL) and H₂O(30 mL) was cooled to 0° C. and added a solution of CAN (86.5 g, 0.48mol) in H₂O (30 mL). The reaction mixture was stirred at roomtemperature for 1 h. The resulting mass was poured into ice cold waterand the aqueous layer was extracted with EtOAc (2×75 mL). The combinedorganic layers were washed with saturated NaHCO₃ followed by brine,dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to get crude. Obtained material was purified by silica gelcolumn chromatography eluting with 70% EtOAc/hexane; finally obtainedmaterial was triturated with 10% EtOAc/hexane to afford 3 (15 g, 36.5%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.99 (d, 1H), 7.42-7.30 (m, 5H), 5.19-5.00(m, 2H), 3.55 (d, 1H), 3.50-3.32 (m, 2H), 3.19 (dd, 1H), 2.18-2.00 (m,2H), 1.91-1.79 (m, 2H)

LCMS m/z: 261 [M⁺+1]

Synthesis of Benzyl 2-(2-ethoxy-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (4)

To a stirred solution of 3 (5 g, 19.23 mmol) in acetonitrile (100 mL)was added CS₂CO₃ (15.6 g, 48 mmol) and ethyl 2-bromoacetate (3.2 mL,28.84 mmol) at RT and stirring was continued for 10 h at RT. Thevolatiles were evaporated under reduced pressure. The residue wasdiluted with water and extracted with EtOAc (2×50 mL). The separatedorganic layer was washed with brine, dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The obtained crude material waspurified by silica gel column chromatography eluting with 30%EtOAc/hexane to afford 4 (3.7 g, 56%) as pale yellow syrup.

¹H-NMR: (500 MHz, CDCl₁): δ 7.38 (d, 5H), 5.19-5.04 (m, 2H), 4.49-3.17(m, 8H), 2.47-2.39 (m, 1H), 2.25-2.21 (m, 1H), 2.05-2.01 (m, 1H),1.95-1.92 (m, 1H), 1.30-1.25 (m, 3H)

LCMS (m/z): 345 [M⁺+1]

Synthesis of 2-(5-((benzyloxy)carbonyl)-1-oxo-2,5-diazaspiro [3.4]octan-2-yl)acetic acid (5)

To a stirred solution of 4 (2 g, 5.783 mmol) in THF: H₂O (24 mL, 5:1)was added LiOH.H₂O (606 mg, 14.4 mmol) at RT and stirred for 4 h. Aftercomplete consumption of the starting material (by TLC), the volatileswere evaporated under reduced pressure. The residue was diluted withwater, washed with ether, the aqueous layer was acidified to pH˜3-4using Citric acid and extracted with EtOAc (2×50 mL). The organic layerswere washed with brine, dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to afford 5 (1.5 g, 83%) as pale yellow syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.41 (d, 5H), 5.07-5.04 (m, 2H), 4.49-3.17(m, 8H), 2.21-2.09 (m, 2H), 1.95-1.92 (m, 2H). LCMS (m/z): 319.4 [M⁺+1]

Synthesis of Benzyl 2-(2-(((2S, 3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl) amino)-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (6)

To a stirring solution of 5 (1 g, 3.14 mmol) in CH₂Cl₂ (50 mL) was addedEDCI (719 mg, 3.76 mmol), DIPEA (2.8 mL, 15.7 mmol) followed by HOBt(635 mg, 3.76 mmol) and Int-F (784 mg, 3.77 mmol) and at 0° C. Thereaction mixture was stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction was quenched with water andextracted with CH₂Cl₂ (2×50 mL). The organic layer was dried overanhydrous Na₂SO₄ and concentrated under vacuum. The crude was purifiedby column chromatography to afford 6 (0.8 g, 57%).

LCMS m/z: 473.4 [M⁺+1]

Synthesis of2-(5-acetyl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)-N-((2S,3R)-3-hydroxy-1-oxo-1-pyrrolidin-1-yl)butan-2-yl)acetamide(Compound I)

To a stirring solution of 6 (0.3 g, 0.63 mmol) in EtOAc (50 mL) wasadded acetic anhydride (0.13 g, 1.27 mmol) followed by Pd/C (0.15 g)under N₂ atmosphere. The reaction mixture was stirred at RT for 16 hunder H₂ atmosphere. After consumption of the starting material (byTLC), the reaction mixture was filtered through a pad of celite andwashed with EtOAc (10 mL). Obtained filtrate was concentrated underreduced pressure to afford crude compound was purified by columnchromatography by eluting 2% MeOH:DCM to afford Compound 1 (0.09 g, 37%)as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.08 (t, J=8.4 Hz, 1H), 4.83-4.75 (m, 1H),4.44-4.38 (m, 1H), 3.93-3.83 (m, 3H), 3.72-3.67 (m, 1H), 3.64-3.61 (m,4H), 3.60-3.58 (m, 1H), 3.57-3.46 (m, 2H), 2.13-2.10 (m, 2H), 2.09 (s,3H), 2.07-1.97 (m, 4H), 1.91-1.87 (m, 2H), 1.04 (d, J=10.8 Hz, 3H)

LCMS m/z: 381.3 [M⁺+1]

HPLC Purity: 46.41 & 47.86 (isomers).

Synthesis of (2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxybutanoicacid (Int-D)

To a stirring solution of (2S,3R)-2-amino-3-hydroxybutanoic acid (Int-C)(10 g, 83.9 mmol) in 1,4-dioxane/water (100 mL, 1:1)) was added NaHCO₃(21.1 g, 0.25 mol) followed by Boc-anhydride (21.9 mL, 0.101 mol) at 0°C. The reaction mixture was stirred at RT for 16 h. After consumption ofthe starting material (by TLC), the reaction mixture was diluted withwater and washed with EtOAc. The aqueous layer was acidified usingcitric acid solution (pH˜3-4) and then extracted with CH₂Cl₂ (2×150 mL).The separated organic extracts were dried over anhydrous Na₂SO₄,filtered and concentrated under vacuum to afford Int-D (15 g, crude).This material was directly used for the next step without furtherpurification.

Synthesis of tert-butyl((253R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)carbamate (Int-E)

To a stirring solution of Int-D (5 g, 22.8 mmol) in CH₂Cl₂ (50 mL) wasadded, EDCI.HCl (5.2 g, 27.3 mmol) HOBt (4.6 g, 34.2 mmol), followed byDIPEA (10.5 mL, 57 mmol) and pyrrolidine (1.945 g, 27.3 mmol) under N₂atmosphere at 0° C. The reaction mixture was stirred at RT for 16 h.After consumption of the starting material (by TLC), the reactionmixture was diluted with DCM and washed with water followed by saturatedNaHCO₃ and citric acid. The separated organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toobtain crude product. This material was purified by columnchromatography eluting with 2% MeOH/DCM to afford Int-E (3 g, 48%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 6.41 (d, 1H), 4.71 (d, 1H), 4.15 (t, 1H),3.94 (q, 1H), 3.63-3.42 (m, 2H), 3.24 (q, 1H), 1.90-1.81 (m, 4H), 1.38(s, 9H), 1.04 (s, 3H)

LCMS m/z: 273.2 [M⁺+1]

Synthesis of (2S,3R)-2-amino-3-hydroxy-1-(pyrrolidin-1-yl)butan-1-one(Int-F)

To a stirring solution of Int-E (3 g, 11.0 mmol) in DCM (10 mL) wasadded ether-HCl (20 mL) at 0° C. under N₂ atmosphere. The reactionmixture was stirred at RT for 4 h. The reaction mixture was concentratedunder reduced pressure to get crude product, which was washed with etherto afford Int-F (2.0 g, 87%).

¹H-NMR: (500 MHz, DMSO-d₅): δ 8.19 (br s, 3H), 3.98-3.91 (m, 2H),3.62-3.59 (m, 1H), 3.49-3.42 (m, 1H), 3.39-3.35 (m, 2H), 1.96-1.90 (m,4H), 1.17 (d, 3H)

LCMS m/z: 173.3 [M⁺+1]

Synthesis of (S)-1-((benzyloxy) carbonyl) pyrrolidine-2-carboxylic acid(Int-A)

To a stirring solution of L-Proline (250 g, 2.17 mol) in water (1 L) wasadded Na₂CO₃ (576 g, 5.43 mol) and stirred for 1 h. After being cooledto 0° C., benzylchloroformate (50% in PhCH₃) (444 g, 2.61 mol) was addeddrop wise to the reaction mixture and again stirred for 1 h. Theresulting reaction mixture was warmed to RT and further stirred for 24h. After consumption of the starting material (by TLC), the reaction wasdiluted with water (1 L) and ether (1.5 L). The separated aqueous layerwas treated with PhCH₃ (1.5 L) and acidified using 6N HCl. The aqueouslayer was extracted with EtOAc (3×1.5 L), combined organic extracts werewashed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford Int-A (450 g, 84%) aslight yellow syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.71 (br s, 1H), 7.37-7.26 (m, 5H),5.07-4.99 (m, 2H), 4.25-4.15 (m, 1H), 3.45-3.34 (m, 2H), 2.25-2.14 (m,1H), 1.94-1.79 (m, 3H)

LCMS m/z: 250.4 [M⁺+1]

Synthesis of Benzyl 2-(chlorocarbonyl)pyrrolidine-1-carboxylate (Int-B)

To a stirring solution of Int-A (100 g, 0.40 mol) in CH₂Cl₂ (500 mL) wasadded catalytic amount of DMF (1 mL) and the reaction mixture was cooledto 0° C. To this oxalyl chloride (112.3 mL, 0.60 mol) was added dropwise and the reaction mixture was stirred at room temperature for 2 h.After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure to afford Int-B (100 g)as crude. This material was directly used for the next step withoutfurther purification.

Example 9—Synthesis of Compound J

Synthesis of 1, 3, 5-Tris (4-methoxybenzyl)-1, 3, 5-triazinane (1)

To a stirring solution of (4-methoxyphenyl) methanamine (SM) (200 g,1.46 mol) in EtOH (600 mL) at room temperature was added formaldehyde(33% aq, 105 mL) drop wise. The reaction mixture was stirred at roomtemperature for 1 h. After consumption of the starting material (byTLC), the reaction mixture was diluted with EtOAc (100 mL) and washedwith water (100 mL) followed by brine. The separated organic layer wasconcentrated under reduced pressure to obtain crude; which was washedwith n-hexane to afford 1 (200 g, 30.6%) as white solid. ¹H-NMR: (500MHz, DMSO-d₆): δ 7.18 (d, 6H), 6.84 (d, 6H), 3.73 (s, 9H), 3.50 (s, 6H),3.29 (s, 6H).

Synthesis of Benzyl 2-(4-methoxybenzyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (2)

To a stirring solution of Int-B (100 g, 0.37 mol) in dry CH₂Cl₂ (500 mL)cooled to −40° C. was added Et₃N (210.2 mL, 1.50 mol) drop wise. Thereaction mixture was stirred at −40° C. for 45 min. To this, a mixtureof compound 1 (50 g, 0.12 mol) and BF₃.OEt₂ (47.6 g, 0.33 mol) in CH₂Cl₂(150 mL) was added drop wise at −40° C. The resulting reaction mixturewas allowed to stir at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was washed with saturated NaHCO₃solution followed by brine. The separated organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The crudematerial was dissolved in EtOAc and kept in the refrigerator forcrystallization. Obtained crystals were filtered and washed with coldEtOAc and dried under vacuum to afford 2 (82 g, 58%) as whitecrystalline solid.

¹H-NMR: (400 MHz, CDCl₃): δ 7.35 (d, 5H), 7.20 (d, 1H), 7.00 (d, 1H),6.85 (d, 1H), 6.75 (d, 1H), 5.15-5.10 (m, 2H), 4.29 (d, 1H), 3.79 (d,3H), 3.59 (d, 1H), 3.57-3.49 (m, 2H), 3.10 (dd, 1H), 2.41-2.30 (m, 1H),2.09-2.00 (m, 2H), 1.70-1.65 (m, 1H), 1.37 (t, 1H)

LCMS m/z: 381 [M⁺+1]

Synthesis of Benzyl 1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate (3)

To a stirring solution of 2 (60 g, 0.16 mol) in MeCN (200 mL) and H₂O(30 mL) was cooled to 0° C. and added a solution of CAN (86.5 g, 0.48mol) in H₂O (30 mL). The reaction mixture was stirred at roomtemperature for 1 h. The resulting mass was poured into ice cold waterand the aqueous layer was extracted with EtOAc (2×75 mL). The combinedorganic layers were washed with saturated NaHCO₃ followed by brine,dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to get crude. Obtained material was purified by silica gelcolumn chromatography eluting with 70% EtOAc/hexane; obtained materialwas triturated with 10% EtOAc/hexane to afford 3 (15 g, 36.5%) as aliquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.99 (d, 1H), 7.42-7.30 (m, 5H), 5.19-5.00(m, 2H), 3.55 (d, 1H), 3.50-3.32 (m, 2H), 3.19 (dd, 1H), 2.18-2.00 (m,2H), 1.91-1.79 (m, 2H)

LCMS m/z: 261 [M⁺+1]

Synthesis of Benzyl 2-(2-ethoxy-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (4)

To a stirred solution of 3 (5 g, 19.23 mmol) in acetonitrile (100 mL)was added Cs₂CO₃ (8.1 g, 24.99 mmol) and ethyl 2-bromoacetate (3.2 mL,28.84 mmol) at RT and stirring was continued for 10 h at RT. Thevolatiles were evaporated under reduced pressure. The residue wasdiluted with water and extracted with EtOAc (2×50 mL). The separatedorganic layer was washed with brine, dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The obtained crude material waspurified by silica gel column chromatography eluting with 20%EtOAc/hexane to afford 4 (4.4 g, 66%) as pale yellow syrup.

¹H-NMR: (500 MHz, CDCl₃): δ 7.38 (d, 5H), 5.19-5.04 (m, 2H), 4.49-3.17(m, 8H), 2.47-2.39 (m, 1H), 2.25-2.21 (m, 1H), 2.05-2.01 (m, 1H),1.95-1.92 (m, 1H), 1.30-1.25 (m, 3H)

LCMS (m/z): 345 [M⁺+1]

Synthesis of Ethyl 2-(5-acetyl-1-oxo-2, 5-diazaspiro [3.4]octan-2-yl)acetate (5)

To a stirring solution of 4 (3.2 g, 9.24 mmol) in EtoAc (20 mL) wasadded acetic anhydride (1.8 g, 17.64 mmol) followed by Pd/C (1.2 g)under N₂ atmosphere. The reaction mixture was stirred at RT for 12 hunder H₂ atmosphere. After consumption of the starting material (byTLC), the reaction mixture was filtered through a pad of celite andwashed with EtoAc (20 mL). Obtained filtrate was concentrated underreduced pressure to afford crude compound was purified by columnchromatography by eluting EtOAc to 5 (2.0 g, 86%) as yellow liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 4.21 (s, 1H), 4.17-4.08 (m, 2H), 3.82 (s,1H), 3.78 (s, 1H), 3.57-3.41 (m, 2H), 3.30-3.29 (m, 1H), 2.12-2.09 (m,2H), 2.07 (s, 3H), 1.92-1.87 (m, 2H), 1.19 (t, J=14.0 Hz, 3H)

LCMS m/z: 255.2 [M⁺+1]

Synthesis of 2-(5-Acetyl-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl)aceticacid (6)

To a stirred solution of 5 (0.65 g, 2.55 mmol) in THF: H₂O (10 mL, 1:1)was added LiOH.H₂O (0.268 g, 6.38 mmol) at RT and stirred for 5 h. Aftercomplete consumption of the starting material (by TLC), the volatileswere evaporated under reduced pressure. The residue was diluted withwater, washed with ether, the aqueous layer was acidified to pH˜2 using2N HCl and extracted with EtOAc (2×20 mL). The organic layers werewashed with brine, dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to afford 6 (0.58 g, 51%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.50 (br s, 1H), 4.12 (s, 1H), 3.72 (s,1H), 3.65 (d, J=4.8 Hz, 1H), 3.55-3.42 (m, 2H), 3.30 (d, J=4.4 Hz, 1H),2.11-2.08 (m, 2H), 1.99 (s, 3H), 1.89-1.86 (m, 2H)

LCMS m/z: 225.1[M⁺−1]

Synthesis of 2-(5-Acetyl-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl)-N-((1S,2R)-2-hydroxy-1-(1, 2, 4-oxadiazol-5-yl) propyl) acetamide (Compound J)

To a stirring solution of 6 (0.5 g, 2.21 mmol) in CH₂Cl₂ (20 mL) wasadded EDCI (0.63 g, 3.31 mmol), HOBt (0.44 g, 3.31 mmol) followed byDIPEA (1.4 g, 10.85 mmol) and Int-I (0.37 g, 2.65 mmol) at 0° C. Thereaction mixture was stirred at RT for 12 h. After consumption of thestarting material (by TLC), the reaction was diluted with water andextracted with CH₂Cl₂ (2×20 mL). The organic layer was dried overanhydrous Na₂SO₄ and concentrated under vacuum. The crude was purifiedby column chromatography by eluting 2% MeOH:DCM to afford Compound J(0.09 g, 12%) as yellow liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.94 (s, 1H), 8.61 (t, J=8.4 Hz, 1H),5.23-5.20 (m, 1H), 5.16-5.09 (m, 1H), 4.20-4.01 (m, 1H), 3.91-3.85 (m,1H), 3.73-3.71 (m, 1H), 3.65 (s, 1H), 3.59-3.57 (m, 1H), 3.49-3.34 (m,1H), 2.16-2.09 (m, 2H), 2.05 (s, 3H), 1.98-1.89 (m, 3H), 1.13-1.11 (m,3H)

LCMS m/z: 352.2 [M⁺+1]

Synthesis of (S)-1-((benzyloxy) carbonyl) pyrrolidine-2-carboxylic acid(Int-A)

To a stirring solution of (S)-pyrrolidine-2-carboxylic acid (250 g, 2.17mol) in water (1 L) was added Na₂CO₃ (576 g, 5.43 mol) and stirred for 1h. After being cooled to 0° C., benzylchloroformate (444 g, 2.61 mol)was added drop wise to the reaction mixture and again stirred for 1 h.The resulting reaction mixture was warmed to RT and further stirred for24 h. After consumption of the starting material (by TLC), the reactionwas diluted with water (1 L) and ether (1.5 L). The separated aqueouslayer acidified using 6N HCl. The aqueous layer was extracted with EtOAc(3×1.5 L); Combined organic extracts were washed with brine, dried overanhydrous Na₂SO₄, filtered and concentrated under vacuum to afford Int A(450 g, 84%) as light yellow syrup.

¹H-NMR: (400 MHz, DMSO-d₆): 12.71 (br s, 1H), 7.40-7.30 (m, 5H),5.19-5.01 (m, 2H), 4.25 (dd, 1H), 3.51-3.50 (m, 2H), 2.29-2.15 (m, 1H),1.89-1.80 (m, 3H)

LCMS m/z: 250 [M⁺+1]

Synthesis of (S)-benzyl 2-(chlorocarbonyl)pyrrolidine-1-carboxylate(Int-B)

To a stirring solution of Int-A (2.5 g, 0.01 mol) in CH₂Cl₂ (50 mL) wasadded SOCl₂ (2.7 g, 0.02 mol) at 0° C. and stirred for 2 h. The reactionmixture was concentrated under reduced pressure to afford Int-B ascrude. This material was directly used for the next step without furtherpurification.

Synthesis of 2-((tert-butoxycarbonyl) amino)-3-hydroxybutanoic acid(Int-D)

To a stirring solution of 2-amino-3-hydroxybutanoic acid (10 g, 83.9mmol) in 1,4-dioxane/water (100 mL, 1:1) was added NaHCO₃ (21.1 g, 0.25mol) followed by Boc-anhydride (21.9 mL, 0.101 mol) at 0° C. Thereaction mixture was stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was diluted with waterand washed with EtOAc. The aqueous layer was acidified using citric acidsolution (pH˜3-4) and then extracted with CH₂Cl₂ (2×150 mL). Theseparated organic extracts were dried over anhydrous Na₂SO₄, filteredand concentrated under vacuum to afford Int-D (15 g, crude). Thismaterial was directly used for the next step without furtherpurification.

Synthesis of 3-(tert-butoxycarbonyl)-2, 2,5-trimethyloxazolidine-4-carboxylic acid (Int-E)

To a stirring solution of Int-D (15 g, 59.28 mmol) in THF (150 mL) wasadded PPTS (1.47 g, 5.92 mmol) followed by 2,2-dimethoxy propane (21.79mL, 0.17 mol) at 0° C. under N₂ atmosphere. The reaction mixture wasstirred at RT for 16 h. The reaction mixture was again heated to refluxfor 6 h. The reaction mixture was diluted with aqueous NaHCO₃ solutionand washed with EtOAc. Aqueous layer was acidified using citric acidsolution (pH˜2) and extracted with CH₂Cl₂ (2×100 mL). The organic layerwas washed with brine, dried over anhydrous Na₂SO₄ and concentratedunder vacuum to afford Int-E (18 g, crude).

¹H-NMR: (400 MHz, DMSO-d₆): δ 13.25 (br s, 1H), 4.11-4.05 (m, 1H), 3.79(d, 1H), 1.50 (s, 3H), 1.67 (s, 3H), 1.45 (s, 9H), 1.29 (d, 3H)

Synthesis of tert-butyl 4-carbamoyl-2, 2,5-trimethyloxazolidine-3-carboxylate (Int-F)

To a stirring solution of Int-E (18 g, 69.4 mmol) in CH₂Cl₂ (180 mL) wasadded, EDCI.HCl (19.88 g, 0.104 mol) HOBt (14.16 g, 0.104 mol), followedby NH₄Cl (5.56 g, 0.104 mol) and DIPEA (31.9 mL, 0.173 mol) at 0° C. Thereaction mixture was stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was washed with aqueouscitric acid, NaHCO₃ followed by brine. Organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to give crude;which was purified by silica gel column chromatography eluting with 2%MeOH/CH₂Cl₂ to afford Int-F (13 g, 72.5%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.51 (br s, 1H), 7.14 (br s, 1H),3.97-3.95 (m, 1H), 3.71 (d, 1H), 1.51 (d, 6H), 1.34 (s, 9H), 1.24 (d,3H)

LCMS (ESI): 159.1[(M⁺+1)-Boc]

Synthesis of (Z)-tert-butyl 4-(((dimethylamino) methylene) carbamoyl)-2,2, 5-trimethyloxazolidine-3-carboxylate (Int-G)

A solution of Int-F (13 g, 50.3 mmol) in DMF.DMA (130 mL) was stirred atreflux temperature for 3 h under N₂ atmosphere. After consumption of thestarting material (by TLC), the reaction mixture was concentrated underreduced pressure to afford Int-G (15.7 g, crude). This crude materialwas directly taken for the next step without further purification.

Synthesis of tert-butyl 2, 2, 5-trimethyl-4-(1, 2, 4-oxadiazol-5-yl)oxazolidine-3-carboxylate (Int-H)

To a stirring solution of Int-G (15.7 g, 50.09 mmol) in ethanol (157 mL)was added hydroxylamine hydrochloride (6.96 g, 0.10 mol) under N₂atmosphere. The reaction mixture was heated to reflux and stirred for 2h. After consumption of the starting material (by TLC), acetic acid(28.6 mL, 0.50 mol) was added to the reaction mixture at RT, and thenrefluxed for 16 h. The solvents from the reaction mixture was evaporatedunder vacuum to give crude; which was purified by silica gel columnchromatography eluting with 10% EtOAc/Hexane to afford Int-H (4.5 g,32%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 6.35 (s, 2H), 4.61 (d, 1H), 4.22-4.15 (m,1H), 1.55 (s, 6H), 1.37 (s, 2H), 1.25 (d, 3H), 1.21 (s, 6H)

LCMS (ESI): 284 [M⁺+1]

Synthesis of 1-amino-1-(1, 2, 4-oxadiazol-5-yl) propan-2-ol (Int-I)

To a stirring solution of Int-H (5 g, 17.6 mmol) in water (25 mL) wasadded trifluoroacetic acid (25 mL). The reaction mixture was stirred atRT for 5 h. After consumption of the starting material (by TLC), thereaction mixture was concentrated under vacuum. The residue wasdissolved in water and neutralized with aqueous NaHCO₃. The solventsfrom the reaction mixture was evaporated under vacuum and extracted with5% MeOH/CH₂Cl₂ (4×150 mL). The organic layer was concentrated underreduced pressure to afford Int-I (2.5 g, crude).

¹H-NMR: (400 MHz, D₂O): δ 8.84 (s, 1H), 4.05 (d, 1H), 3.98-3.95 (m, 1H),3.67 (s, 1H), 3.58 (d, 1H), 1.15 (d, 3H), 1.12 (d, 3H)

LCMS (ESI): 144.1 [M⁺+1].

Example 10—Synthesis of Compound K

Synthesis of 1, 3, 5-Tris (4-methoxybenzyl)-1, 3, 5-triazinane (1)

To a stirring solution of (4-methoxyphenyl) methanamine SM (200 g, 1.46mol) in EtOH (600 mL) at room temperature was added formaldehyde (33%aq, 105 mL) drop wise. The reaction mixture was stirred at roomtemperature for 1 h. After consumption of the starting material (byTLC), the reaction mixture was diluted with EtOAc (100 mL) and washedwith water (100 mL) followed by brine. The separated organic layer wasconcentrated under reduced pressure to obtain crude; which was washedwith n-hexane to afford 1 (200 g, 30.6%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.18 (d, J=8.0 Hz, 6H), 6.81 (d, J=8.0 Hz,6H), 3.71 (s, 9H), 3.50 (s, 6H), 3.29 (s, 6H)

Synthesis of Benzyl 2-(4-methoxybenzyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (2)

To a stirring solution of 1 (45 g, 100 mmol) in CH₂Cl₂ (150 mL) wasadded BF₃.OEt₂ (37 mL, 301 mmol) drop wise at −40° C. To above stirringsolution Int-B (95 g, 342 mmol) in dry CH₂Cl₂ (500 mL) followed by Et₃N(210.2 mL, 1.50 mol) was drop wise. The reaction mixture was stirred at−40° C. for 45 min. The resulting reaction mixture was allowed to stirat RT for 16 h. After consumption of the starting material (by TLC), thereaction mixture was washed with saturated NaHCO₃ solution followed bybrine. The separated organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The crude material was dissolved inEtOAc and kept in the refrigerator for crystallization. Obtainedcrystals were filtered and washed with cold EtOAc and dried under vacuumto afford 2 (90 g, 65%) as white crystalline solid.

¹H-NMR: (500 MHz, DMSO-d6): 7.36-7.30 (m, 5H), 7.24 (d, J=8.0 Hz, 1H),7.06 (d, J=8.0 Hz, 1H), 6.90 (d, J=7.5 Hz, 1H), 6.81 (d, J=8.5 Hz, 1H),5.09 (s, 2H), 4.29 (s, 1H), 4.13, 3.96 (dd, J=15.5 Hz, 15.0 Hz, 1H),3.73 (s, 3H), 3.11 (t, J=5.0 Hz, 2H), 2.16-2.09 (m, 2H), 1.83-1.77 (m,2H), 1.20-1.15 (m, 2H)

LCMS m/z: 381 [M⁺+1]

Synthesis of Benzyl 1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate (3)

A stirring solution of 2 (46 g, 121 mmol) in MeCN (460 mL) and H₂O (200mL) were cooled to 0° C. and added a solution of CAN (199 g, 0.23 mol)in H₂O (460 mL). The reaction mixture was stirred at room temperaturefor 1 h. The resulting mass was poured into ice cold water (100 mL) andthe aqueous layer was extracted with EtOAc (2×200 mL). The combinedorganic layers were washed with saturated NaHCO₃ followed by brine,dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to afford crude material was purified by silica gel columnchromatography eluting with EtOAc to obtained 3 (12 g, 38%) as anoff-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.90 (s, 1H), 7.36-7.29 (m, 5H), 5.10 (s,2H), 3.53 (d, 0.1=4.5 Hz, 2H), 3.36-3.30 (m, 1H), 3.17, 3.13 (dd, J=5.0Hz, 5.0 Hz, 1H), 2.17-2.10 (m, 2H), 1.82-1.76 (m, 2H)

LCMS m/z: 261 [M⁺+1]

Synthesis of Benzyl 2-(2-ethoxy-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (4)

To a stirred solution of 3 (12 g, 46.1 mmol) in acetonitrile (120 mL)was added Cs₂CO₃ (37.6 g, 115.2 mmol) and ethyl 2-bromoacetate (7.7 mL,69.2 mmol) at RT and stirred for 16 h at RT. After completion ofreaction (by TLC), the volatiles were evaporated under reduced pressure.The residue was diluted with water (50 mL) and extracted with EtOAc(2×100 mL). The separated organic layer was washed with brine, driedover anhydrous Na₂SO₄ and concentrated under reduced pressure. Theobtained crude material was purified by silica gel column chromatographyeluting with 80% EtOAc/hexane to afford 4 (12.5 g, 78.6%) as pale brownsyrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.35-7.30 (m, 5H), 5.06 (s, 2H), 4.21 (s,1H), 4.18 (s, 1H), 4.13-4.10 (m, 2H), 3.69 (d, J=4.5 Hz, 1H), 3.47-3.44(m, 3H), 2.16 (t, J=6.0 Hz, 2H), 1.87-1.80 (m, 2H), 1.21-1.14 (m, 3H)

LCMS m/z: 369.3 [M⁺+Na]

Synthesis of ethyl 2-(5-acetyl-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl)acetate (5)

To a stirring solution of 4 (12.5 g, 36 mmol) in EtOAc (100 mL) wereadded acetic anhydride (7.36 g, 72.2 mmol), 50% wet 10% Pd/C (5.0 g) andstirred under H₂ atmosphere (balloon pressure) for 4 h at RT. Aftercompletion of reaction (by TLC), the reaction mixture was filteredthrough a pad of celite and triturated with EtOAc (50 mL). The filtratewas concentrated under reduced pressure to afford 5 (8.0 g, 87.9%) asyellow syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 4.21 (s, 1H), 4.17 (s, 1H), 4.14-4.12 (m,1H), 3.82 (s, 1H), 3.68 (d, J=4.8 Hz, 1H), 3.56-3.51 (m, 1H), 3.46-3.43(m, 1H), 3.29 (d, J=4.8 Hz, 2H), 2.11-2.09 (m, 1H), 1.97 (s, 2H),1.90-1.89 (m, 3H), 1.20 (t, J=7.2 Hz, 3H).

Synthesis of 2-(5-acetyl-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl) aceticacid (6)

To a stirred solution of 5 (8.0 g, 31.49 mmol) in THF: H₂O (80 mL/30 mL)were added LiOH.H₂O (3.30 g, 78.7 mmol) at RT and stirred for 2 h. Afterconsumption of the starting material (by TLC), the volatiles wereevaporated under reduced pressure. The residue was diluted with water(25 mL), extracted with ether (2×50 mL). The separated aqueous layer wasacidified to pH˜2 using 2N HCl and extracted with 5% MeOH/DCM (3×50 mL).The organic layers were dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to afford 6 (6.5 g, 91.5%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.5 (br s, 1H), 4.12 (s, 1H), 3.70 (s,1H), 3.66-3.64 (m, 2H), 3.53-3.51 (m, 2H), 3.53-3.51 (m, 2H), 3.42 (d,J=7.0 Hz, 1H), 2.73, 2.63 (dd, J=16.0 Hz, J=15 Hz, 2H), 2.10-2.07 (m,1H)

LCMS m/z: 227.2 [M⁺+1]

Synthesis of 2-(5-acetyl-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl)-N-((1S,2R)-2-hydroxy-1-(1, 3, 4-oxadiazol-2-yl) propyl) acetamide (Compound K)

To a stirring solution of 6 (200 mg, 1.39 mmol) in DCM (10 mL) wereadded AT, N-diisopropylethylamine (0.64 mL, 3.47 mmol), Int-I (5.95 g,1.68 mmol), followed by HATU (637 mg, 1.68 mmol) at 0° C. and stirred atRT for 16 h. After consumption of the starting material (by TLC), thereaction mixture was concentrated under reduced pressure to give crudeproduct, which was purified by column chromatography by 8% MeOH/DCM toafford yellow syrup which was further purified by preparative HPLC toafford Compound K (100 mg, 20.4%) as colorless liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.18 (d, J=3.6 Hz, 1H), 8.54 (t, J=7.6 Hz,1H), 5.15-5.06 (m, 2H), 4.16-4.12 (m, 1H), 4.05, 4.00 (dd, J=4.4 Hz, 4.4Hz, 1H), 3.89-3.82 (m, 1H), 3.72 (t, J=5.6 Hz, 1H), 3.61-3.56 (m, 1H),3.51-3.45 (m, 1H), 3.37-3.33 (m, 1H), 2.18-2.08 (m, 2H), 2.02 (s, 3H),1.93-1.90 (m, 2H), 1.10 (t, J=6.4 Hz, 3H)

LCMS m/z: 352.3 [M⁺+1]

UPLC: 47.2% & 44.3%

Synthesis of (S)-1-((benzyloxy) carbonyl) pyrrolidine-2-carboxylic acid(Int-A)

To a stirring solution of L-proline (250 g, 2.17 mol) in water (1 L) wasadded Na₂CO₃ (576 g, 5.43 mol) and stirred for 1 h. After being cooledto 0° C., benzylchloroformate (50% in PhCH₃) (444 g, 2.61 mol) was addeddrop wise to the reaction mixture and again stirred for 1 h. Theresulting reaction mixture was warmed to RT and further stirred for 24h. After consumption of the starting material (by TLC), the reaction wasdiluted with water (1 L) and ether (1.5 L). The separated aqueous layerwas treated with PhCH₃ (1.5 L) and acidified with 6N HCl. The aqueouslayer was extracted with EtOAc (3×1.5 L), combined organic extracts werewashed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford Int-A (450 g, 84%) as paleyellow syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.71 (br s, 1H), 7.37-7.26 (m, 5H),5.07-4.99 (m, 2H), 4.25-4.15 (m, 1H), 3.45-3.34 (m, 2H), 2.25-2.14 (m,1H), 1.94-1.79 (m, 3H)

LCMS m/z: 250.4 [M⁺+1]

Synthesis of (S)-benzyl 2-(chlorocarbonyl) pyrrolidine-1-carboxylate(Int-B)

To a stirring solution of Int-A (2.5 g, 0.01 mol) in CH₂Cl₂ (50 mL) wasadded SOCl₂ (2.7 g, 0.02 mol) at 0° C. and stirred for 2 h. The reactionmixture was concentrated under reduced pressure to afford Int-B ascrude. This material was directly used for the next step without furtherpurification.

Synthesis of (2S, 3R)-methyl 2-amino-3-hydroxybutanoate (Int-C)

To a stirring solution of L-Thr-OH (60 g, 504 mmol) in CH₃OH (400 mL)was added thionyl chloride (70 mL, 972 mmol) at 0° C. and stirred at 75°C. for 6 h. After completion of starting material (by TLC), the reactionmixture was concentrated under reduced pressure to afford Int-C (60 g,crude). This material was directly used for the next step withoutfurther purification.

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.45 (s, 2H), 5.70 (s, 1H), 4.12-4.10 (m,1H), 3.90 (s, 1H), 3.73 (s, 3H), 1.20 (d, J=6.5 Hz, 3H).

Synthesis of (2S, 3R)-methyl 2-(((benzyloxy) carbonyl)amino)-3-hydroxybutanoate (Int-D)

To a stirring solution of NaHCO₃ (89 g, 1.065 mol) in water/1,4-dioxane(150 mL/450 mL) were added Int-C (60 g, 355 mmol) at RT and stirred for30 min. The reaction mixture was cooled to 0° C. Cbz-Cl (60.7 mL, 426mmol) was added drop wise and stirred for 1 h. The reaction mixture wasstirred to RT and stirred for 16 h. After completion of startingmaterial (by TLC), the reaction mass was diluted with EtOAc (300 ml).The separated organic layer was washed with (2×200 mL) of saturatedNaHCO₃ solution followed by brine solution (2×100 mL). The organic layerwas dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to afford crude material which was triturated with n-hexane anddiethylether (50 mL/50 mL) to afford Int-D (60 g, 63.8%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.37-7.30 (m, 5H), 7.20 (d, J=8.4 Hz, 1H),5.06 (s, 2H), 4.78 (d, J=6.8 Hz, 1H), 4.09-4.05 (m, 2H), 3.64 (s, 3H),1.09 (d, J=6.0 Hz, 3H)

LCMS m/z: 268.2[M⁺+1]

Synthesis of (2S, 3R)-methyl 2-(((benzyloxy) carbonyl)amino)-3-((tort-butyldimethylsilyl) oxy) butanoate (Int-E)

To a stirring solution of Int-D (40 g, 149 mmol) in DMF (300 mL) wereadded DIPEA (69 mL, 374 mmol), TBDMS-Cl (30.91 mL, 179 mmol) at 0° C.and stirred at RT for 16 h. After completion of starting material (byTLC), the reaction mass was diluted with ether (200 ml). The separatedorganic layer was washed with (2×200 mL) of saturated NaHCO₃ solutionfollowed by brine solution (2×100 mL). The organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toafford crude material was purified by column chromatography eluting 10%EtOAc/hexane to afford Int-E (40 g, 70.1%) as colorless syrup.

¹H-NMR: (400 MHz, CDCl₃): δ 7.39-7.32 (m, 5H), 5.43 (d, J=9.6 Hz, 1H),5.14 (s, 2H), 4.45-4.43 (m, 1H), 4.29-4.26 (m, 1H), 3.72 (s, 3H), 1.21(d, J=6.0 Hz, 3H), 0.83 (s, 9H), 0.09 (s, 6H)

LCMS m/z: 382.2[M⁺+1].

Synthesis of benzyl ((2S, 3R)-3-((tert-butyldimethylsilyl)oxy)-1-hydrazinyl-1-oxobutan-2-yl) carbamate (Int F)

A solution of Int-E (20 g, 52.4 mmol) in EtOH (200 mL) was addedhydrazine hydrate (13.12 mL, 262 mmol), at RT and stirred at 90° C. for16 h. After completion of starting material (by TLC), ethanol wasevaporated under reduced pressure. The crude residue was diluted withwater (100 mL) and diethyl ether (200 mL). After the separated organiclayer was dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure. Obtained crude material was purified by columnchromatography by eluting with 15% EtOAc/hexane to afford Int-F (4.0 g,20%) as colorless thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.10 (s, 1H), 7.36-7.30 (m, 5H), 6.83 (d,0.1=9.6 Hz, 1H), 5.02 (s, 2H), 4.19 (s, 2H), 4.05-4.02 (m, 1H),3.97-3.93 (m, 1H), 1.05 (d, J=6.0 Hz, 3H), 0.81 (s, 9H), 0.01 (s, 6H)

Synthesis of benzyl ((1S, 2R)-2-((tert-butyldimethylsilyl) oxy)-1-(1, 3,4-oxadiazol-2-yl) propyl) carbamate (Int-G)

A solution of Int-F (4 g, 10.4 mmol) in triethyl orthoformate (40 mL)was added p-TSA (catalytic, 40 mg) at RT and after stirred at 120° C.for 3 h. After completion of starting material (by TLC),triethylorthoformate was evaporated under reduced pressure. The cruderesidue was purified by column chromatography eluting 10% EtOAc/hexaneto afford Int-G (2.8 g, 68%) as white solid.

¹H-NMR: (500 MHz, DMSO-d6): δ 9.22 (s, 1H), 7.85 (d, J=9.5 Hz, 1H),7.36-7.31 (m, 5H), 5.05 (s, 2H), 4.96-4.93 (m, 1H), 4.25 (t, J=6.0 Hz,1H), 1.23 (d, J=6.0 Hz, 3H), 0.80 (s, 9H), 0.10 (s, 6H)

LCMS m/z: 392.4[M⁺+1]

Synthesis of (1S, 2R)-2-((tert-butyldimethylsilyl) oxy)-1-(1, 3,4-oxadiazol-2-yl) propan-1-amine (Int-H)

To a stirring solution of Int-G (2.8 g, 7.16 mmol) in methanol (30 mL)was added 50% wet 10% Pd/C (1.4 g) and stirred under H₂ atmosphere(balloon pressure) for 2 h at RT. The reaction mixture was filteredthrough a pad of celite and triturated with methanol (10 mL). Thefiltrate was concentrated under reduced pressure to afford Int-H (1.7 g,92%) as colorless syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.15 (s, 1H), 4.11 (t, J=5.0 Hz, 1H), 4.03(d, J=2.0 Hz, 1H), 2.05 (br s, 2H), 1.17 (d, J=6.0 Hz, 3H), 0.76 (s,9H), 0.02 (s, 6H)

LCMS m/z: 258.3 [M⁺+1]

Synthesis of (1S, 2R)-1-amino-1-(1, 3, 4-oxadiazol-2-yl) propan-2-ol(Int-I)

To a stirring solution of Int-H (500 mg, 1.94 mmol) in THF (6 mL) wasadded TBAF (1.01 mL) slowly at 0° C. and stirred at RT for 3 h. Aftercompletion of reaction (by TLC), the reaction mixture was evaporated anddiluted with EtOAc/H₂O (10 mL/2 mL). The separated organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to afford Int-1 (120 mg, crude) as colorless thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.12 (s, 1H), 3.94 (d, J=4.5 Hz, 1H), 3.85(t, J=5.5 Hz, 1H), 3.17-3.13 (m, 3H), 1.05 (d, J=6.0 Hz, 3H).

Example 11—Synthesis of Compound L

Synthesis of 1, 3, 5-Tris (4-methoxybenzyl)-1, 3, 5-triazinane (1)

To a stirring solution of (4-methoxyphenyl) methanamine SM (200 g, 1.46mol) in EtOH (600 mL) at room temperature was added formaldehyde (33%aq, 105 mL) drop wise. The reaction mixture was stirred at roomtemperature for 1 h. After consumption of the starting material (byTLC), the reaction mixture was diluted with EtOAc (100 mL) and washedwith water (100 mL) followed by brine. The separated organic layer wasconcentrated under reduced pressure to obtain crude; which was washedwith n-hexane to afford 1 (200 g, 30.6%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.18 (d, J=8.0 Hz, 6H), 6.81 (d, J=8.0 Hz,6H), 3.71 (s, 9H), 3.50 (s, 6H), 3.29 (s, 6H)

Synthesis of Benzyl 2-(4-methoxybenzyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (2)

To a stirring solution of 1 (45 g, 100 mmol) in CH₂Cl₂ (150 mL) wasadded BF₃.OEt₂ (37 mL, 301 mmol) drop wise at −40° C. Int-B (95 g, 342mmol) in dry CH₂Cl₂ (500 mL) was added, followed by Et₃N (210.2 mL, 1.50mol) drop wise. The reaction mixture was stirred at −40° C. for 45 min.The resulting reaction mixture was allowed to stir at RT for 16 h. Afterconsumption of the starting material (by TLC), the reaction mixture waswashed with saturated NaHCO₃ solution followed by brine. The separatedorganic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The crude material was dissolved in EtOAc and kept inthe refrigerator for crystallization. Obtained crystals were filteredand washed with cold EtOAc and dried under vacuum to afford 2 (90 g,65%) as white crystalline solid.

¹H-NMR: (500 MHz, DMSO-d6): 7.36-7.30 (m, 5H), 7.24 (d, J=8.0 Hz, 1H),7.06 (d, J=8.0 Hz, 1H), 6.90 (d, J=7.5 Hz, 1H), 6.81 (d, J=8.5 Hz, 1H),5.09 (s, 2H), 4.29 (s, 1H), 4.13, 3.96 (dd, J=15.5 Hz, 15.0 Hz, 1H),3.73 (s, 3H), 3.11 (t, J=5.0 Hz, 2H), 2.16-2.09 (m, 2H), 1.83-1.77 (m,2H), 1.20-1.15 (m, 2H)

LCMS m/z: 381 [M⁺+1]

Synthesis of Benzyl 1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate (3)

To a stirring solution of 2 (46 g, 121 mmol) in MeCN (460 mL) and H₂O(200 mL) were cooled to 0° C. and added a solution of CAN (199 g, 0.23mol) in H₂O (460 mL). The reaction mixture was stirred at roomtemperature for 1 h. The resulting mass was poured into ice cold water(100 mL) and the aqueous layer was extracted with EtOAc (2×200 mL). Thecombined organic layers were washed with saturated NaHCO₁ followed bybrine, dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure to afford crude material was purified by silica gelcolumn chromatography eluting with EtOAc to obtain 3 (12 g, 38%) as anoff-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.90 (s, 1H), 7.36-7.29 (m, 5H), 5.10 (s,2H), 3.53 (d, J=4.5 Hz, 2H), 3.36-3.30 (m, 1H), 3.17, 3.13 (dd, J=5.0Hz, 5.0 Hz, 1H), 2.17-2.10 (m, 2H), 1.82-1.76 (m, 2H)

LCMS m/z: 261 [M⁺+1]

Synthesis of Benzyl 2-(2-ethoxy-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (4)

To a stirred solution of 3 (12 g, 46.1 mmol) in acetonitrile (120 mL)was added CS₂CO₁ (37.6 g, 115.2 mmol) and ethyl 2-bromoacetate (7.7 mL,69.2 mmol) at RT and stirred for 16 h at RT. After completion ofreaction (by TLC), the volatiles were evaporated under reduced pressure.The residue was diluted with water (50 mL) and extracted with EtOAc(2×100 mL). The separated organic layer was washed with brine, driedover anhydrous Na₂SO₄ and concentrated under reduced pressure. Theobtained crude material was purified by silica gel column chromatographyeluting with 80% EtOAc/hexane to afford 4 (12.5 g, 78.6%) as pale brownsyrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.35-7.30 (m, 5H), 5.06 (s, 2H), 4.21 (s,1H), 4.18 (s, 1H), 4.13-4.10 (m, 2H), 3.69 (d, J=4.5 Hz, 1H), 3.47-3.44(m, 3H), 2.16 (t, J=6.0 Hz, 2H), 1.87-1.80 (m, 2H), 1.21-1.14 (m, 3H)

LCMS m/z: 369.3 [M⁺+Na].

Synthesis of ethyl 2-(5-acetyl-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl)acetate (5)

To a stirring solution of 4 (12.5 g, 36 mmol) in EtOAc (100 mL) wereadded acetic anhydride (7.36 g, 72.2 mmol), 50% wet 10% Pd/C (5.0 g) andstirred under H₂ atmosphere (balloon pressure) for 4 h at RT. Aftercompletion of reaction (by TLC), the reaction mixture was filteredthrough a pad of celite and triturated with EtOAc (50 mL). The filtratewas concentrated under reduced pressure to afford 5 (8.0 g, 87.9%) asyellow syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 4.21 (s, 1H), 4.17 (s, 1H), 4.14-4.12 (m,1H), 3.82 (s, 1H), 3.68 (d, J=4.8 Hz, 1H), 3.56-3.51 (m, 1H), 3.46-3.43(m, 1H), 3.29 (d, J=4.8 Hz, 2H), 2.11-2.09 (m, 1H), 1.97 (s, 2H),1.90-1.89 (m, 3H), 1.20 (t, J=7.2 Hz, 3H)

LCMS m/z: 255 [M⁺−1]

Synthesis of 2-(5-acetyl-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl) aceticacid (6)

To a stirred solution of 5 (8.0 g, 31.49 mmol) in THF: H₂O (80 mL/30 mL)were added LiOH.H₂O (3.30 g, 78.7 mmol) at RT and stirred for 2 h. Afterconsumption of the starting material (by TLC), the volatiles wereevaporated under reduced pressure. The residue was diluted with water(25 mL), extracted with ether (2×50 mL). The separated aqueous layer wasacidified to pH˜2 using 2N HCl and extracted with 5% MeOH/DCM (3×50 mL).The organic layers were dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to afford 6 (6.5 g, 91.5%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.5 (br s, 1H), 4.12 (s, 1H), 3.70 (s,1H), 3.66-3.64 (m, 2H), 3.53-3.51 (m, 2H), 3.53-3.51 (m, 2H), 3.42 (d,J=7.0 Hz, 1H), 2.73, 2.63 (dd, J=16.0 Hz, J=15 Hz, 2H), 2.10-2.07 (m,1H)

LCMS m/z: 227.2 [M⁺+1]

Synthesis of 2-(5-acetyl-1-oxo-2, 5-diazaspiro [3.4]octan-2-yl)-N-(2-((tert-butyldimethylsilyl) oxy)-1-(pyrimidin-2-yl)propyl) acetamide (7)

To a stirring solution of 6 (400 mg, 1.77 mmol) in DCM (20 mL) wereadded N N-diisopropylethylamine (0.9 mL, 5.32 mmol), Int-I (474 mg, 1.77mmol), HOBt (410 mg, 2.66 mmol), EDCI.HCl (509 mg, 2.66 mmol) at 0° C.and stirred at RT for 16 h. After consumption of the starting material(by TLC), the reaction mixture was concentrated under reduced pressureto give crude product, which was purified by column chromatography by 4%MeOH/DCM to afford 7 (300 mg, 35%) as colorless thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.78 (d, J=16.0 Hz, 2H), 8.35 (d, J=9.0Hz, 1H), 7.37 (d, J=16.0 Hz, 1H), 4.88 (t, J=8.0 Hz, 1H), 4.37 (d, J=8.0Hz, 1H), 3.90 (d, J=8.0 Hz, 2H), 3.85 (s, 2H), 3.76 (d, J=10.0 Hz, 1H),3.63-3.57 (m, 1H), 3.49-3.43 (m, 1H), 3.13 (d, J=9.0 Hz, 1H), 2.13-2.10(m, 2H), 2.01 (s, 3H), 1.16 (d, 0.1=5.5 Hz, 3H), 0.64 (s, 9H), 0.06 (s,6H)

LCMS m/z: 474.6 [M⁺−1]

Synthesis of 2-(5-acetyl-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl)-N-((1R,2R)-2-hydroxy-1-(pyrimidin-2-yl) propyl) acetamide (Compound L)

To a stirring solution of 7 (300 mg, 0.63 mmol) in THF (20 mL) was addedTBAF (1.26 mL) slowly at 0° C. and stirred at RT for 2 h. Aftercompletion of reaction (by TLC), the reaction mixture was evaporated togive crude product, which was purified by column chromatography eluting4% MeOH/DCM to afford mixture (110 mg) of isomers again purified bychiral preparative HPLC method purification to afford Compound L (60 mg,26%) as colorless liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.76 (t, J=6.0 Hz, 2H), 8.28 (d, J=8.8 Hz,1H), 7.37 (t, J=4.8 Hz, 1H), 4.91-4.79 (m, 2H), 4.15-4.10 (m, 1H),3.92-3.78 (m, 2H), 3.67 (d, J=4.8 Hz, 1H), 3.60-3.54 (m, 1H), 3.49-3.31(m, 2H), 2.13-2.11 (m, 2H), 2.09 (s, 3H), 2.08-1.87 (m, 2H), 1.09 (d,J=6.4 Hz, 3H)

LCMS m/z: 362.4 [M⁺+1]

HPLC: 97.5%

Synthesis of (S)-1-((benzyloxy) carbonyl) pyrrolidine-2-carboxylic acid(Int-A)

To a stirring solution of L-proline (250 g, 2.17 mol) in water (1 L) wasadded Na₂CO₃ (576 g, 5.43 mol) and stirred for 1 h. After being cooledto 0° C., benzylchloroformate (50% in PhCH₃) (444 g, 2.61 mol) was addeddrop wise to the reaction mixture and again stirred for 1 h. Theresulting reaction mixture was warmed to RT and further stirred for 24h. After consumption of the starting material (by TLC), the reaction wasdiluted with water (1 L) and ether (1.5 L). The separated aqueous layerwas treated with PhCH₃ (1.5 L) and acidified with 6N HCl. The aqueouslayer was extracted with EtOAc (3×1.5 L), combined organic extracts werewashed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford Int-A (450 g, 84%) as paleyellow syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.71 (br s, 1H), 7.37-7.26 (m, 5H),5.07-4.99 (m, 2H), 4.25-4.15 (m, 1H), 3.45-3.34 (m, 2H), 2.25-2.14 (m,1H), 1.94-1.79 (m, 3H)

LCMS m/z: 250.4 [M⁺+1]

Synthesis of (S)-benzyl 2-(chlorocarbonyl) pyrrolidine-1-carboxylate(Int-B)

To a stirring solution of Int-A (2.5 g, 0.01 mol) in CH₂Cl₂ (50 mL) wasadded SOCl₂ (2.7 g, 0.02 mol) at 0° C. and stirred for 2 h. The reactionmixture was concentrated under reduced pressure to afford Int-B. Thismaterial was directly used for the next step without furtherpurification.

Synthesis of 1-(pyrimidin-2-yl) propan-1-one (Int-D)

To a stirring solution of Int-C (20 g, 190 mmol) in THF (200 mL) wasadded ethyl magnesium bromide (1M in THF, 227 mL, 228 mmol) at 0° C. for1 h. After completion of starting material (by TLC), the reactionmixture was diluted with saturated ammonium chloride solution and EtOAc(150 mL). The separated organic layer was washed with brine solution(2×100 mL). The extracted organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to afford crudematerial which was purified by column chromatography eluting 20%EtOAc/hexane to afford Int-D (15 g, 57%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.00 (d, J=5.2 Hz, 2H), 7.70 (t, J=4.8 Hz,1H), 3.20-3.15 (m, 2H), 1.09 (t, J=7.2 Hz, 3H)

LCMS m/z: 137 [M⁺+1]

Synthesis of (Z)-2-(1-((triethylsilyl) oxy) prop-1-en-1-yl) pyrimidine(Int-E)

To a stirring solution of Int-D (15 g, 110 mmol) in THF (100 mL) wasadded LiHMDS (1M in THF, 220 mL, 220 mmol) slowly at 0° C. and stirredfor 30 min. Then chloro triethylsilane (24.8 g, 165 mmol) in THF (50 mL)was added dropwise at 0° C. and stirred 1 h. After completion ofstarting material (by TLC), the reaction mixture was diluted withsaturated ammonium chloride solution (50 mL) and EtOAc (150 mL). Theseparated organic layer was extracted with brine solution (2×100 mL).The separated organic layer was dried over anhydrous Na₂SO₄, filteredand concentrated under reduced pressure to afford crude material whichwas purified by column chromatography eluting 5% EtOAc/hexane to affordInt-E (20 g, 74%) as yellow thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.75 (d, J=4.8 Hz, 2H), 7.32 (t, J=4.8 Hz,1H), 6.36-6.31 (m, 1H), 1.77 (d, J=7.2 Hz, 3H), 0.95-0.87 (m, 9H),0.71-0.65 (m, 6H).

Synthesis of 2-bromo-1-(pyrimidin-2-yl) propan-1-one (Int-F)

To a stirring solution of Int-E (20 g, 80 mmol) in THF/H₂O (160 mL/40mL) were added N-bromosuccinimide (10.2 g, 88 mmol) slowly at RT andstirred for 2 h. After completion of starting material (by TLC), thereaction mixture was diluted with H₂O and EtOAc (100 ml/150 mL). Theseparated organic layer was extracted with brine solution (2×100 mL).The separated organic layer was dried over anhydrous Na₂SO₄, filteredand concentrated under reduced pressure to afford crude material whichwas purified by column chromatography eluting 30% EtOAc/hexane to affordInt-F (15 g, 87%) as yellow thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.06 (d, J=4.8 Hz, 2H), 7.75 (t, J=4.8 Hz,1H), 5.97-5.92 (m, 1H), 1.83 (d, J=6.4 Hz, 3H).

Synthesis of 2-hydroxy-1-(pyrimidin-2-yl) propan-1-one (Int-G)

To a stirring solution of Int-F (15 g, 69 mmol) in MeOH (240 mL) wasadded sodium formate (18.9 g, 279 mmol) and stirred the reaction mass at70° C. for 8 h. After completion of reaction (by TLC), the reactionmixture was evaporated under reduced pressure to give crude product,which was purified by column chromatography eluting 5% MeOH/DCM toafford Int G (5.5 g, 51%) as colorless liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.73 (d, J=5.2 Hz, 2H), 7.55 (t, J=4.8 Hz,1H), 5.28-5.26 (m, 1H), 1.24 (d, J=6.4 Hz, 1H), 0.99 (d, J=6.4 Hz, 3H)

Synthesis of (2-((tert-butyldimethylsilyl) oxy)-1-(pyrimidin-2-yl)propan-1-one (Int-H)

To a stirring solution of Int-G (5.5 g, 36 mmol) in DCM (150 mL) wereadded imidazole (4.9 g, 72 mmol), DMAP (880 mg, 0.72 mmol) at 0° C. andstirred for 10 min. After added TBDMS-Cl (8.1 g, 54 mmol) at 0° C. andstirred at RT for 6 h. After completion of starting material (by TLC)reaction mixture was diluted with H₂O (50 ml). The separated organiclayer was washed with brine solution (2×50 mL). The organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to afford crude material which was purified by columnchromatography eluting 30% EtOAc/hexane to afford Int-H (3 g, 31%) as anoff-white solid.

¹H-NMR: (400 MHz, CDCl₃): δ 9.00 (d, J=5.2 Hz, 2H), 7.71 (t, J=4.8 Hz,1H), 5.47-5.42 (m, 1H), 1.35 (d, 0.1=6.8 Hz, 3H), 0.79 (s, 9H), 0.05 (s,6H)

Synthesis of (2-((tert-butyldimethylsilyl) oxy)-1-(pyrimidin-2-yl)propan-1-amine (Int-I)

To a stirring solution of Int-H (3 g, 11.2 mmol) in MeOH (50 mL) wereadded sodium acetate (1.8 g, 22.5 mmol), ammonium carbonate (8.8 g, 56.3mmol), AcOH (0.6 mL, 11.2 mmol) at RT and then stirred the reactionmixture at 70° C. for 2 h. The temperature of the reaction was cooled toRT and sodium cyanoborohydride (1.39 g, 22.5 mmol) was added and stirredat 70° C. for 6 h. After completion of starting material (by TLC), MeOHwas evaporated and the crude residue was diluted with DCM/H₂O (50 ml/50mL). The separated organic layer was extracted with brine solution (2×50mL). The organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford crude material waspurified by column chromatography eluting 5% MeOH/DCM to afford Int-I(2.4 g, 80%) as semi solid.

¹H-NMR: (400 MHz, CDCl₃): δ 8.83 (d, J=4.8 Hz, 2H), 7.40 (t, J=5.2 Hz,1H), 4.13 (t, J=6.4 Hz, 2H), 3.90 (d, J=6.4 Hz, 2H), 1.12 (d, J=6.4 Hz,3H), 0.70 (s, 9H), 0.02 (s, 6H)

LCMS m/z: 268[M⁺+1]

Synthesis of (2S, 3R)-2-(((benzyloxy) carbonyl) amino)-3-hydroxybutanoicacid (6S-A)

To a stirring solution of NaHCO₃ (529 g, 6.30 mot) in water (1 L) wasadded L-threonine (250 g, 2.10 mol) at RT and stirred for 30 min. Thereaction mixture was cooled to 0° C. Cbz-Cl (850 mL, 2.52 mol, 50% ofPhCH₃) was added drop wise and stirred for 1 h. The reaction mixture waswarmed to RT and again stirred for 28 h. To this MTBE (1 L) was addedand stirred for 20 min. Separated aqueous layer in toluene was stirredfor 20 min. Aqueous layer was acidified with 1N HCl (pH˜1-2) andextracted with EtOAc (3×1.5 L). The organic layer was washed with brine,dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Thecrude material was stirred with dicyclohexylamine (819 mL, 4.20 mol) for4 h to get white solid, filtered and dried. Obtained solid was refluxedwith EtOAc (1.5 L) for 1 h and then filtered. The solid material wasdissolved in water (1 L) and acidified with dil.H₂SO₄ and again stirredfor 30 min. The aqueous layer was extracted with EtOAc (3×1 L). Theseparated organic layer was washed with brine, dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure. Obtained crudematerial was triturated with n-hexane to afford 6S-A (230 g, 43%) aswhite solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.55 (br m, 1H), 7.37-7.30 (m, 5H), 6.94(d, J=8.8 Hz, 1H), 5.05 (s, 2H), 4.08-3.94 (m, 2H), 1.02 (d, J=6.4 Hz,3H).

ELSD purity: 84.66%

Synthesis of benzyl ((2S, 3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)carbamate (6S-B)

A solution of 6S-A (25 g, 98.8 mmol) in DCM (250 mL) was added ammoniumchloride (7.86 g, 147 mmol), HATU (45 g, 118 mmol), N,N-diisopropylamine (45.5 mL, 261 mmol) and stirred at RT for 16 h. Aftercompletion of starting material (by TLC), the organic layer was washedby saturated sodium bicarbonate solution (1×150 mL) followed by 2N HCl(1×100 mL). After the separated organic layer was dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure. Obtained crudematerial was purified by column chromatography by eluting with 2%MeOH/DCM to afford 6S-B (16 g, 66%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.36-7.32 (m, 5H), 7.04 (s, 1H), 7.25 (s,1H), 5.03 (s, 2H), 4.75 (d, J=6.0 Hz, 1H), 3.95-3.92 (m, 1H), 3.86-3.83(m, 1H), 1.27-1.23 (m, 1H), 1.04 (d, J=6.5 Hz, 3H).

LCMS m/z: 253.3[M⁺+1]

Synthesis of (2R, 3S)-4-amino-3-(((benzyloxy) carbonyl)amino)-4-oxobutan-2-yl acetate (6S-C)

To a stirring solution of 6S-B (16 g, 63.4 mmol) in CH₂Cl₂ (250 mL) wereadded Et₃N (10.5 mL, 76.0 mmol) and DMAP (773 mg, 6.34 mmol), Ac₂O (7.12mL, 76.0 mmol) at 0° C. and stirred at RT for 2 h. After completion ofstarting material (by TLC), the organic layer was washed with water(1×150 mL) followed by brine (1×100 mL) washing. After the separatedorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure. Obtained crude material was purified by columnchromatography by eluting with 1% MeOH/DCM to afford 6S-C (15 g, 80.3%)as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.45 (s, 1H), 7.35-7.30 (m, 5H), 7.24 (d,J=9.2 Hz, 1H), 7.17 (s, 1H), 5.09-5.05 (m, 1H), 5.01 (s, 2H), 4.14-4.10(m, 1H), 1.93 (s, 3H), 1.14 (d, J=6.4 Hz, 3H)

LCMS m/z: 295.1 [M⁺+1]

Synthesis of (2R, 3S)-3, 4-diamino-4-oxobutan-2-yl acetate (6S-D)

To a stirring solution of 6S-C (15 g, 51 mmol) in methanol (500 mL) wasadded 50% wet 10% Pd/C (4 g) and stirred under H₂ atmosphere (balloonpressure) for 4 h at RT. The reaction mixture was filtered through a padof celite and triturated with methanol (50 mL). The filtrate wasconcentrated under reduced pressure to afford 6S-D (7.5 g, 91.9%) as anoff-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.59 (d, J=8.8 Hz, 1H), 7.16 (s, 1H), 7.01(s, 1H), 4.78 (d, J=5.2 Hz, 1H), 4.10 (m, 1H), 4.00-3.96 (m, 1H), 1.89(s, 3H), 1.01 (d, J=6.4 Hz, 3H)

LCMS m/z: 161.5 [M⁺+1]

Synthesis of (2S,3R)-methyl 2-amino-3-hydroxybutanoate (6S-E)

To a stirring solution of (2S, 3R)-2-amino-3-hydroxybutanoic acid (200g, 1.68 mol) in methanol (1.2 L) was added SOCl₂ (244 mL, 3.36 mol)dropwise at 0° C. and stirred for 1 h. The resulting reaction mixturewas refluxed for 24 h. After consumption of the starting material (byTLC), the reaction mixture was warmed to RT and concentrated undervacuum and decanted with n-hexane (2×50 mL). The residue was dissolvedin EtOH (1 L) and neutralized with Et₃N (471 mL, 3.36 mol) and againstirred for 2 h. The precipitated solid was filtered off; obtainedfiltrate was concentrated under vacuum to afford 6S-E (195 g, 80%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.51 (br s, 3H), 4.13-4.10 (m, 1H), 3.91(br s, 1H), 1.20 (d, 3H).

LCMS m/z: 134.1 [M⁺+1]

Synthesis of (2S, 3R)-2-amino-3-hydroxybutanamide (6S-F)

A solution of 6S-E (190 g, 1.35 mol) in IPA (2 L) was taken in autoclaveand purged NH₃ gas (7-8 kg) and stirred at 35° C. for 24 h. Aftercompletion of the reaction, NH₃ was expelled and reaction mixture wasconcentrated under reduced pressure and added CH₂Cl₂ and filtered.Obtained solid was refluxed in EtOH for 1 h at 78° C. The reaction masswas filtered in heating condition and n-hexane was added to the filtrateand again stirred for another 4 h. Obtained precipitated solid wasfiltered and dried under vacuum to afford 6S-F (160 g, 47%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.38 (br s, 1H), 7.02 (br s, 1H), 4.66 (brs, 1H), 3.77-3.70 (m, 1H), 2.93 (d, 1H), 2.72 (br m, 1H), 1.05 (d, 3H).

LCMS 119.1[M⁺+1].

Synthesis of (S)-1-((benzyloxy) carbonyl) pyrrolidine-2-carboxylic acid(6S-G)

To a stirring solution of L-proline (250 g, 2.17 mol) in water (1 L) wasadded Na₂CO₃ (576 g, 5.43 mol) and stirred for 1 h. After being cooledto 0° C., benzylchloroformate (50% in PhCH₃) (444 g, 2.61 mol) was addeddrop wise to the reaction mixture and again stirred for 1 h. Theresulting reaction mixture was warmed to RT and further stirred for 24h. After consumption of the starting material (by TLC), the reaction wasdiluted with water (1 L) and ether (1.5 L). The separated aqueous layerwas treated with PhCH₃ (1.5 L) and acidified using 6N HCl. The aqueouslayer was extracted with EtOAc (3×1.5 L), combined organic extracts werewashed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford 6S-G (450 g, 84%) as lightyellow syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.71 (br s, 1H), 7.37-7.26 (m, 5H),5.07-4.99 (m, 2H), 4.25-4.15 (m, 1H), 3.45-3.34 (m, 2H), 2.25-2.14 (m,1H), 1.94-1.79 (m, 3H).

LCMS m/z: 250.4 [M⁺+1]

Synthesis of (S)-benzyl 2-(chlorocarbonyl) pyrrolidine-1-carboxylate(6S-H)

To a stirring solution of 6S-G (90 g, 361 mmol) in CH₂Cl₂ (400 mL) wasadded oxalyl chloride (42 mL, 542 mmol) at 0° C. and stirred for 2 h.After complete formation of acid chloride, the reaction mixture wasconcentrated under reduced pressure to afford 6S-H (95 g, crude). Thismaterial was directly used for the next step without furtherpurification.

1, 3, 5-Tris (4-methoxybenzyl)-1, 3, 5-triazinane (6S-I)

To a stirring solution of (4-methoxyphenyl) methenamine (200 g, 1.46mol) in EtOH (600 mL) at room temperature was added formaldehyde (33%aq, 105 mL) dropwise. The reaction mixture was stirred at roomtemperature for 1 h. After consumption of the starting material (byTLC), the reaction mixture was diluted with EtOAc (100 mL) and washedwith water (100 mL) followed by brine. The separated organic layer wasconcentrated under reduced pressure to obtain crude; which was finallywashed with n-hexane to afford compound 6S-I (200 g, 30.6%) as whitesolid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.18 (d, J=8.0 Hz, 6H), 6.81 (d, J=8.0 Hz,6H), 3.71 (s, 9H), 3.50 (s, 6H), 3.29 (s, 6H).

Benzyl 2-(4-methoxybenzyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (6S-J)

To a stirring solution of 6S-H (100 g, 0.37 mol) in dry CH₂Cl₂ (500 mL)was cooled to −40° C. and added Et₃N (210.2 mL, 1.50 mol) dropwise. Thereaction mixture was stirred at −40° C. for 45 min. To this a mixture of6S-I (50 g, 0.12 mol) in CH₂Cl₂ (150 mL) and BF₃.OEt₂ (47.6 g, 0.33 mol)was added drop wise at −40° C. The resulting reaction mixture wasallowed to stir at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was washed with saturated NaHCO₃solution followed by brine. The separated organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The crudematerial was dissolved in EtOAc and kept in the refrigerator forcrystallization. Obtained crystals were filtered and washed with coldEtOAc and dried under vacuum to afford 6S-J (82 g, 58%) as whitecrystalline solid.

¹H-NMR: (500 MHz, DMSO-d6): δ 7.36-7.30 (m, 5H), 7.24 (d, J=8.0 Hz, 1H),7.06 (d, J=8.0 Hz, 1H), 6.90 (d, J=7.5 Hz, 1H), 6.81 (d, J=8.5 Hz, 1H),5.09 (s, 2H), 4.29 (s, 1H), 4.13-3.96 (m, 1H), 3.73 (s, 3H), 3.11 (t,J=5.0 Hz, 2H), 2.16-2.09 (m, 2H), 1.83-1.77 (m, 2H), 1.20-1.15 (m, 2H).

LCMS m/z: 381 [M⁺+1]

Benzyl 1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate (6S-K)

To a stirring solution of 6S-J (30 g, 78.94 mmol) in MeCN (300 mL) andH₂O (150 mL) were cooled to 0° C. and added a solution of CAN (129 g,0.23 mol) in H₂O (300 mL). The reaction mixture was stirred at roomtemperature for 1 h. The resulting mass was poured into ice cold waterand the aqueous layer was extracted with EtOAc (2×150 mL). The combinedorganic layers were washed with saturated NaHCO₃ followed by brine,dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to get crude. Obtained material was purified by silica gelcolumn chromatography eluting with EtOAc to afford 6S-K (8 g, 40%) as anoff-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.90 (s, 1H), 7.36-7.29 (m, 5H), 5.10 (s,2H), 3.53 (d, J=4.5 Hz, 2H), 3.36-3.30 (m, 1H), 3.17-3.13 (m, 1H),2.17-2.10 (m, 2H), 1.82-1.76 (m, 2H).

LCMS m/z: 261 [M⁺+1]

Benzyl 2-(2-ethoxy-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (6S-L)

To a stirred solution of 6S-K (10.0 g, 38.46 mmol) in acetonitrile (100mL) were added CS₂CO₃ (31.34 g, 96.19 mmol) and ethyl 2-bromoacetate(6.42 mL, 57.60 mmol) at RT and stirred for 16 h at RT. The volatileswere evaporated under reduced pressure. The residue was diluted withwater and extracted with EtOAc (2×100 mL). The separated organic layerwas washed with brine, dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure. The obtained crude material was purified bysilica gel column chromatography eluting with 80% EtOAc/Hexane to afford6S-L (10.0 g, 75%) as pale brown syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.35-7.30 (m, 5H), 5.06 (s, 2H), 4.21 (s,1H), 4.18 (s, 1H), 4.13-4.10 (m, 2H), 3.69 (d, J=4.5 Hz, 1H), 3.47-3.44(m, 3H), 2.16 (t, J=6.0 Hz, 2H), 1.87-1.80 (m, 2H), 1.21-1.14 (m, 3H).

LCMS m/z: 261 [M⁺+1]

2-(5-((benzyloxy) carbonyl)-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl)acetic acid (6S-M)

To a stirred solution of 6S-L (6.0 g, 17.34 mmol) in THF: H₂O (75 mL/40mL) were added LiOH.H₂O (1.82 g, 43.33 mmol) at RT and stirred for 2 h.After consumption of the starting material (by TLC), the volatiles wereevaporated under reduced pressure. The residue was diluted with water,washed with ether, the aqueous layer was acidified to pH˜2 using 2N HCland extracted with EtOAc (2×50 mL). The organic layers were washed withbrine, dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to afford compound to afford 6S-M (4.5 g, 88.2%) as anoff-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.5 (br s, 1H), 7.35-7.30 (m, 5H), 5.06(s, 2H), 4.21 (s, 1H), 4.18 (s, 1H), 3.69 (d, J=4.5 Hz, 1H), 3.47-3.44(m, 3H), 2.16 (t, J=6.0 Hz, 2H), 1.87-1.80 (m, 2H)

LCMS m/z: 319 [M⁺+1]

Synthesis of 1-tert-butyl 2-methyl pyrrolidine-1,2-dicarboxylate (6S-N)

To a stirring solution of proline methyl ester (70 g, 422 mmol) inCH₂Cl₂ (700 mL) were added Et₃N (183 mL, 1.26 mol) at 0° C. and stirredfor 10 min. After added Boc-anhydride (184 mL, 845 mmol) at 0° C. andthe reaction mixture was stirred at RT for 16 h. After consumption ofthe starting material (by TLC), the reaction was diluted with water (200mL) and extracted with CH₂Cl₂ (2×200 mL). The combined organic layer waswashed with citric acid (1×150 mL), brine (1×200 mL). The organic layerwas dried over Na₂SO₄ and concentrated under reduced pressure to affordcrude compound which was purified by column chromatography by eluting50% EtOAc/n-hexane to obtain 6S-N (80 g, 83%) as thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 4.15-4.13 (m, 1H), 3.65 (s, 3H), 3.35-3.30(m, 2H), 2.23-2.15 (m, 1H), 1.86-1.78 (m, 3H), 1.41 (s, 9H);

LCMS (m/z): 129 [(M⁺+1)-Boc];

Synthesis of 1-tert-butyl 2-methyl2-((benzyloxy)methyl)pyrrolidine-1,2-dicarboxylate (6S-O)

To a stirring solution of 6S-N (25 g, 109 mmol) in THF (250 mL) wasadded LiHMDS (240 mL, 240 mmol) at −20° C. and stirred for 2 h. To thisBOM-chloride (23 mL, 163 mmol) was added drop wise at −30° C. andstirred for 2 h. After consumption of the starting material (by TLC),the reaction was quenched with aqueous NH₄Cl solution (100 mL) andextracted with EtOAc (2×200 mL). The combined organic layer was washedwith water (2×150 mL) followed by brine solution (2×100 mL). The organiclayer was dried over Na₂SO₄ and concentrated to obtain crude compoundwhich was purified by column chromatography by eluting 10%EtOAc/n-hexane to afford 6S-O (30 g, 79%) as thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.36-7.22 (m, 5H), 4.59-4.48 (m, 2H),4.02-3.88 (m, 1H), 3.63 (s, 3H), 3.49-3.35 (m, 2H), 3.34-3.30 (m, 1H),2.31-2.23 (m, 1H), 2.04-1.89 (m, 2H), 1.82-1.78 (m, 1H);

LCMS (m/z): 249.4 [(M⁺+1)-Boc]

Synthesis of2-((benzyloxy)methyl)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylicacid (6S-P)

To a stirring solution of 6S-O (30 g, 86 mmol) in methanol (70 mL) wasadded NaOH solution (6.88 g in 70 mL H₂O) at RT. The reaction mixturewas heated to 70° C. for 16 h. After consumption of the startingmaterial (by TLC), the solvent from the reaction was evaporated underreduced pressure and diluted with EtOAc (2×200 mL). The separatedaqueous layer was acidified using citric acid solution (pH′-3) andextracted with EtOAc (2×250 mL). The combined organic layer was driedover Na₂SO₄ and concentrated to afford crude was triturated withn-hexane to obtain 6S-P (25 g, 86.8%) as off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ12.35 (br s, 1H), 7.37-7.29 (m, 5H),4.56-4.48 (m, 2H), 4.06-4.00 (m, 1H), 3.92-3.89 (m, 1H), 3.66-3.45 (m,1H), 3.37-3.28 (m, 1H), 2.31-2.20 (m, 1H), 2.05-1.97 (m, 1H), 1.87-1.75(m, 2H), 1.38 (s, 9H)

LCMS (m/z): 335.3 [M⁺+1]

Synthesis of1-(tert-butoxycarbonyl)-2-(hydroxymethyl)pyrrolidine-2-carboxylic acid(6S-Q)

To a stirring solution of 6S-P (25 g, 74 mmol) in methanol (150 mL) wasadded 50% wet 10% Pd/C (7 g) at RT and stirred for 10 h under H₂atmosphere. After consumption of the starting material (by TLC), thereaction mixture was filtered through a pad of celite and the pad waswashed with methanol (100 mL). Obtained filtrate was concentrated underreduced pressure to afford 6S-Q (15 g, 82.8%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ4.66 (br s, 1H), 3.96-3.83 (m, 1H),3.63-3.59 (m, 1H), 3.49-3.41 (m, 1H), 3.34-3.25 (m, 1H), 2.30-2.17 (m,1H), 1.95-1.72 (m, 3H), 1.38 (s, 9H).

Synthesis oftert-butyl2-(hydroxymethyl)-2-((2-methoxy-2-oxoethyl)carbamoyl)pyrrolidine-1-carboxylate(6S-R)

To a stirring solution of 6S-Q (25 g, 102 mmol) in CH₂Cl₂ (250 mL) wereadded DIPEA (54.3 mL, 303 mmol), EDCI (23.2 g, 122 mmol), HOBT (16.5 g,122 mmol), glycine methyl ester.HCl (15.3 g, 122 mmol) at 0° C. andstirred to RT for 12 h. After consumption of the starting material (byTLC), the reaction mixture was diluted with water (100 mL) and extractedwith CH₂Cl₂ (2×100 mL). The combined organic layer was washed withcitric acid (1×100 mL) followed by bicarbonate solution (1×100 mL). Theorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure. Obtained crude material was purified by silicagel column chromatography eluting with 20% EtOAc/n-hexane to afford 6S-R(20 g, 62.5%) as yellow thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.02 (d, J=19.5 Hz, 1H), 5.01 (t, J=5.0Hz, 1H), 4.03-4.00 (m, 3H), 3.84-3.76 (m, 2H), 3.71-3.68 (m, 1H), 3.61(s, 3H), 2.27-2.23 (m, 2H), 1.98-1.75 (m, 2H), 1.38 (s, 9H);

Mass (ESI): m/z 217.2 [M⁻-Boc+1]

Synthesis of tert-butyl2-(2-methoxy-2-oxoethyl)-1-oxo-2,5-diazaspiro[3.4]octane-5-carboxylate(6S-S)

To a stirring solution of triphenylphosphine (72.5 g, 276 mmol) in THF(200 mL) was added DIAD (54.5 g, 276 mmol) at RT and stirred for 30 min.After added 6S-R (35 g, 110 mmol) in (60 mL) THF slowly and reactionmixture was stirred at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction was concentrated under reduced pressure.The crude material was triturated with 30% diethyl ether/n-hexane (2×500mL). The filtered organic solvent was concentrated and crude residue waspurified by column chromatography by eluting 30% EtOAc/n-hexane toafford 6S-S (30 g, crude) as semi solid.

¹H-NMR: (500 MHz, CDCl₃): δ4.50-4.37 (m, 1H), 3.86 (s, 3H), 3.74-3.63(m, 2H), 3.47-3.31 (m, 1H), 3.69-3.60 (m, 1H), 3.42-3.33 (m, 2H),3.28-3.23 (m, 1H), 2.18-2.14 (m, 2H), 1.90-1.76 (m, 2H), 1.40 (s, 9H)

Mass (ESI): m/z 319.3 [M⁺+1]

Synthesis of2-(5-(tert-butoxycarbonyl)-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)aceticacid (6S-T)

To a stirring solution of 6S-S (10 g, 33.5 mmol) in THF/H₂O (100 mL)were added LiOH.H₂O (2.11 g, 50.2 mmol) at RT and stirred for 16 h.After consumption of the starting material (by TLC), reaction mass wasevaporated under reduced pressure. The crude was washed with diethylether (2×200 mL). The separated aqueous layer was acidified by usingcitric acid solution (pH˜3) and extracted with EtOAc (3×100 mL). Thecombined organic layer was dried over Na₂SO₄ and concentrated to afford6S-T (5 g, 52.6%)

¹H-NMR: (500 MHz, DMSO-d₆): δ12.80 (br s, 1H), 4.14-4.10 (m, 1H),3.74-3.54 (m, 2H), 3.38-3.33 (m, 2H), 3.30-3.23 (m, 1H), 2.14-2.08 (m,2H), 1.83-1.76 (m, 2H), 1.39 (s, 9H);

LCMS (m/z): 283.3 [M⁻−1],

HPLC: 97.6%

(2S, 3R)-2-((tert-butoxycarbonyl) amino)-3-hydroxybutanoic acid (6S-U)

To a stirring solution of (2S, 3R)-2-amino-3-hydroxybutanoic acid (10 g,83.9 mmol) in 1, 4-dioxane/water (100 mL, 1:1)) was added NaHCO₃ (21.1g, 0.25 mol) followed by Boc-anhydride (21.9 mL, 0.101 mol) at 0° C. Thereaction mixture was stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was diluted with waterand washed with EtOAc. The aqueous layer was acidified using citric acidsolution (pH˜3-4) and then extracted with CH₂Cl₂ (2×150 mL). Theseparated organic extracts were dried over anhydrous Na₂SO₄, filteredand concentrated under reduced pressure to afford 6S-U (15 g, crude).This material was directly used for the next step without furtherpurification.

tert-butyl((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)carbamate (6S-V)

To a stirring solution of 6S-U (5 g, 22.8 mmol) in CH₂Cl₂ (50 mL) wasadded EDCI.HCl (5.2 g, 27.3 mmol), HOBt (4.6 g, 34.2 mmol) followed byDIPEA (10.5 mL, 57 mmol) and pyrrolidine (1.945 g, 27.3 mmol) under N₂atmosphere at 0° C. The reaction mixture was stirred at RT for 16 h.After consumption of the starting material (by TLC), the reactionmixture was diluted with DCM and washed with water followed by saturatedNaHCO₃ and citric acid. The separated organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toobtain crude product. This material was purified by columnchromatography eluting with 2% MeOH/DCM to afford 6S-V (3 g, 48%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 6.41 (d, 1H), 4.71 (d, 1H), 4.15 (t, 1H),3.94 (q, 1H), 3.63-3.42 (m, 2H), 3.24 (q, 1H), 1.90-1.81 (m, 4H), 1.38(s, 9H), 1.04 (s, 3H).

LCMS (m/z): 273.2 [M⁺+1]

(2S,3R)-2-amino-3-hydroxy-1-(pyrrolidin-1-yl)butan-1-one (6S-W)

To a stirred solution of 6S-V (3 g, 11.0 mmol) in DCM (10 mL) was addeddiethyl ether saturated with HCl (20 mL) at 0° C. under N₂ atmosphere.The reaction mixture was stirred at RT for 4 h. The reaction mixture wasconcentrated under reduced pressure to get crude product, which waswashed with ether to afford 6S-W (2.0 g, 87%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.19 (br s, 3H), 3.98-3.91 (m, 2H),3.62-3.59 (m, 1H), 3.49-3.42 (m, 1H), 3.39-3.35 (m, 2H), 1.96-1.90 (m,4H), 1.17 (d, 3H).

LCMS (m/z): 173.3 [M⁺+1]

Synthesis of (2S, 3R)-methyl 2-amino-3-hydroxybutanoate (6S-X)

To a stirring solution of L-threonine (50 g, 420 mmol) in CH₃OH (250 mL)was added thionyl chloride (62.4 mL, 840 mmol) at 0° C. and stirred at75° C. for 6 h. After completion of starting material (by TLC), thereaction mixture was concentrated under reduced pressure to afford 6S-X(60 g, crude). This material was directly used for the next step withoutfurther purification.

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.45 (s, 2H), 5.70 (s, 1H), 4.12-4.10 (m,1H), 3.90 (s, 1H), 3.73 (s, 3H), 1.20 (d, J=6.5 Hz, 3H).

Synthesis of (2S 3R)-methyl 2-(((benzyloxy) carbonyl)amino)-3-hydroxybutanoate (6S-Y)

To a stirring solution of 6S-X (60 g, 353 mmol) in water/1,4 dioxane(150 mL/300 mL) The reaction mixture was cooled to 0° C. added NaHCO₃(88.9 g, 1.059 mol) at 0 C and stirred for 15 min. Cbz-Cl (60.7 mL, 426mmol) was added drop wise and stirred for 1 h. The reaction mixture wasstirred to RT and stirred for 12 h. After completion of startingmaterial (by TLC), diluted the reaction mass with EtOAc (300 ml). Theseparated organic layer was washed with (2×200 mL) of saturated NaHCO₃solution followed by brine solution (2×100 mL). The organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to afford crude material was triturated with n-hexane (500 mL)to afford 6S-Y (70 g, 74%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.37-7.30 (m, 5H), 7.20 (d, J=8.4 Hz, 1H),5.06 (s, 2H), 4.78 (d, J=6.8 Hz, 1H), 4.09-4.05 (m, 2H), 3.64 (s, 3H),1.09 (d, J=6.0 Hz, 3H).

LCMS m/z: 268.2[M⁺+1]

Synthesis of (2S, 3R)-methyl 2-(((benzyloxy) carbonyl)amino)-3-((tort-butyldimethylsilyl) oxy) butanoate (6S-Z)

To a stirring solution of 6S-Y (50 g, 187 mmol) in DMF (400 mL) wereadded DIPEA (86 mL, 468 mmol) TBDMS-Cl (33.66 mL, 224 mmol) at 0° C. andstirred at RT for 12 h. After completion of starting material (by TLC),diluted the reaction mass with EtOAc (500 ml). The separated organiclayer was washed with (2×200 mL) of H₂O followed by brine solution(2×100 mL). The organic layer was dried over anhydrous Na₂SO₄, filteredand concentrated under reduced pressure to afford crude material waspurified by column chromatography eluting 10% EtOAc/hexane to afford6S-Z (50 g, 70.1%) as colorless syrup.

¹H-NMR: (400 MHz, CDCl₃): δ 7.39-7.32 (m, 5H), 5.43 (d, J=9.6 Hz, 1H),5.14 (s, 2H), 4.45-4.43 (m, 1H), 4.29-4.26 (m, 1H), 3.72 (s, 3H), 1.21(d, J=6.0 Hz, 3H), 0.83 (s, 9H), 0.09 (s, 6H) LCMS m/z: 382.2 [M⁺+1]

Synthesis of benzyl ((2S, 3R)-3-((tert-butyldimethylsilyl)oxy)-1-hydrazinyl-1-oxobutan-2-yl) carbamate (6S-AA)

A solution of 6S-Z (50 g, 131 mmol) in EtOH (400 mL) was added hydrazinehydrate (32.8 g, 656 mmol), at RT and after stirred at 90° C. for 24 h.After completion of starting material (by TLC), ethanol was evaporatedunder reduced pressure. The crude residue was diluted with water (100mL) and EtOAc (500 mL). After the separated organic layer was washedwith (2×100 mL) of Water followed by brine solution (1×100 mL). driedover anhydrous Na₂SO₄, filtered and concentrated under reduced pressure.Obtained crude material was purified by column chromatography by elutingwith 20% EtOAc/hexane to afford 6S-AA (25 g, 50%) as colorless thicksyrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.10 (s, 1H), 7.36-7.30 (m, 5H), 6.83 (d,J=9.6 Hz, 1H), 5.02 (s, 2H), 4.19 (s, 2H), 4.05-4.02 (m, 1H), 3.97-3.93(m, 1H), 1.05 (d, J=6.0 Hz, 3H), 0.81 (s, 9H), 0.01 (s, 6H).

Synthesis of benzyl ((1S, 2R)-2-((tert-butyldimethylsilyl) oxy)-1-(1, 3,4-oxadiazol-2-yl) propyl) carbamate (6S-AB)

A solution of 6S-AA (25 g, 65.6 mmol) in triethyl orthoformate (250 mL)was added p-TSA (catalytic, 250 mg) at RT and after stirred at 80° C.for 3 h. After completion of starting material (by TLC), triethylorthoformate was evaporated under reduced pressure. The crude residuewas purified by column chromatography eluting 10% EtOAc/hexane to afford6S-AB (15 g, 58%) as thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.22 (s, 1H), 7.85 (d, J=9.5 Hz, 1H),7.36-7.31 (m, 5H), 5.05 (s, 2H), 4.96-4.93 (m, 1H), 4.25 (t, J=6.0 Hz,1H), 1.23 (d, J=6.0 Hz, 3H), 0.80 (s, 9H), 0.10 (s, 6H);

LCMS m/z: 392.4 [M⁺+1]

Synthesis of (1S, 2R)-2-((tert-butyldimethylsilyl) oxy)-1-(1, 3,4-oxadiazol-2-371) propan-1-amine (6S-AC)

To a stirring solution of 6S-AB (15 g, 38.3 mmol) in methanol (200 mL)was added 50% wet 10% Pd/C (5 g) and stirred under H₂ atmosphere(balloon pressure) for 4 h at RT. The reaction mixture was filteredthrough a pad of celite and triturated with methanol (100 mL). Thefiltrate was concentrated under reduced pressure to afford 6S-AC (10 g,crude) as thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.15 (s, 1H), 4.11 (t, J=5.0 Hz, 1H), 4.03(d, J=2.0 Hz, 1H), 2.05 (br s, 2H), 1.17 (d, J=6.0 Hz, 3H), 0.76 (s,9H), 0.02 (s, 6H)

LCMS m/z: 258.3 [M⁺+1]

Synthesis of (1S, 2R)-1-amino-1-(1, 3, 4-oxadiazol-2-yl) propan-2-ol(6S-AD)

To a stirring solution of 6S-AC (10 g, 38.9 mmol) in THF (100 mL) wasadded TBAF (20.3 g, 77.8 mmol) slowly at 0° C. and stirred at RT for 3h. After completion of reaction (by TLC), the reaction mixture wasevaporated and diluted with EtOAc/H₂O (200 mL/50 mL). The separatedorganic layer was dried over anhydrous N₂SO₄, filtered and concentratedunder reduced pressure. The crude residue was purified by columnchromatography eluting 5% MeOH/DCM to afford 6S-AD (5 g, crude) as thicksyrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.12 (s, 1H), 3.94 (d, J=4.5 Hz, 1H), 3.85(t, J=5.5 Hz, 1H), 3.17-3.13 (m, 3H), 1.05 (d, J=6.0 Hz, 3H).

LCMS m/z: 144 [M⁺+1]

Synthesis of 2-((tert-butoxycarbonyl) amino)-3-hydroxybutanoic acid(6S-AE)

To a stirring solution of 2-amino-3-hydroxybutanoic acid (10 g, 83.9mmol) in 1,4-dioxane/water (100 mL, 1:1)) were added NaHCO₃ (21.1 g,0.25 mol) followed by Boc-anhydride (21.9 mL, 0.101 mol) at 0° C. Thereaction mixture was stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was diluted with waterand washed with EtOAc. The aqueous layer was acidified using citric acidsolution (pH˜3-4) and then extracted with CH₂Cl₂ (2×150 mL). Theseparated organic extracts were dried over anhydrous Na₂SO₄, filteredand concentrated under vacuum to afford 6S-AE (15 g, crude). Thismaterial was directly used for the next step without furtherpurification.

Synthesis of 3-(tert-butoxycarbonyl)-2, 2,5-trimethyloxazolidine-4-carboxylic acid (6S-AF)

To a stirring solution of 6S-AE (15 g, 59.28 mmol) in THF (150 mL) wasadded PPTS (1.47 g, 5.92 mmol) followed by 2,2-dimethoxy propane (21.79mL, 0.17 mol) at 0° C. under N₂ atmosphere. The reaction mixture wasstirred at RT for 16 h. The reaction mixture was again heated to refluxfor 6 h. The reaction mixture was diluted with aqueous NaHCO₃ solutionand washed with EtOAc (1×100 mL). Aqueous layer was acidified usingcitric acid solution (pH˜2) and extracted with CH₂Cl₂ (2×100 mL). Theorganic layer was washed with brine, dried over anhydrous Na₂SO₄ andconcentrated under vacuum to afford 6S-AF (18 g, crude).

¹H-NMR: (400 MHz, DMSO-d₆): δ13.25 (br s, 1H), 4.11-4.05 (m, 1H), 3.79(d, 1H), 1.50 (s, 3H), 1.67 (s, 3H), 1.45 (s, 9H), 1.29 (d, 3H).

Synthesis of tert-butyl 4-carbamoyl-2, 2,5-trimethyloxazolidine-3-carboxylate (6S-AG)

To a stirring solution of 6S-AF (18 g, 69.4 mmol) in CH₂Cl₂ (180 mL) wasadded HOBt (14.16 g, 0.104 mol), EDCI.HCl (19.88 g, 0.104 mol) followedby NH₄Cl (5.56 g, 0.104 mol) and DIPEA (31.9 mL, 0.173 mol) at 0° C. Thereaction mixture was stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was washed with aqueouscitric acid, NaHCO₃ followed by brine. Organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to give crude;which was purified by silica gel column chromatography eluting with 2%MeOH/CH₂Cl₂ to afford 6S-AG (13 g, 72.5%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.51 (br s, 1H), 7.14 (br s, 1H),3.97-3.95 (m, 1H), 3.71 (d, 1H), 1.51 (d, 6H), 1.34 (s, 9H), 1.24 (d,3H).

LCMS (ESI): 159.1[(M⁺+1)-Boc]

Synthesis of (Z)-tert-butyl 4-(((dimethylamino) methylene) carbamoyl)-2,2, 5-trimethyloxazolidine-3-carboxylate (6S-AH)

A solution of 6S-AG (13 g, 50.3 mmol) in DMF.DMA (130 mL) was stirred atreflux temperature for 3 h under N₂ atmosphere. After consumption of thestarting material (by TLC), the reaction mixture was concentrated underreduced pressure to afford 6S-AH (15.7 g, crude). This crude materialwas directly taken for the next step without further purification.

Synthesis of tert-butyl 2, 2, 5-trimethyl-4-(1, 2, 4-oxadiazol-5-yl)oxazolidine-3-carboxylate (6S-AI)

To a stirring solution of 6S-AH (15.7 g, 50.09 mmol) in ethanol (157 mL)was added hydroxylamine hydrochloride (6.96 g, 0.10 mol) under N₂atmosphere. The reaction mixture was heated to reflux and stirred for 2h. After consumption of the starting material (by TLC), acetic acid(28.6 mL, 0.50 mol) was added to the reaction mixture and then refluxedfor 16 h. The solvents from the reaction mixture was evaporated undervacuum to give crude; which was purified by silica gel columnchromatography eluting with 10% EtOAc/Hexane to afford 6S-AI (4.5 g,32%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 6.35 (s, 2H), 4.61 (d, 1H), 4.22-4.15 (m,1H), 1.55 (s, 6H), 1.37 (s, 2H), 1.25 (d, 3H), 1.21 (s, 6H).

LCMS (ESI): 284 [M⁺+1]

Mass (m/z): 283 [M⁺]

Synthesis of 1-amino-1-(1, 2, 4-oxadiazol-5-yl) propan-2-ol (6S-AJ)

To a stirring solution of 6S-AI (5 g, 17.6 mmol) in water (25 mL) wasadded trifluoroacetic acid (25 mL). The reaction mixture was stirred atRT for 5 h. After consumption of the starting material (by TLC), thereaction mixture was concentrated under vacuum. The residue wasdissolved in water and neutralized with aqueous NaHCO₃. The solvent fromthe reaction mixture was evaporated under vacuum and extracted with 5%MeOH/CH₂Cl₂ (3×100 mL). The organic layer was concentrated under reducedpressure to afford 6S-AJ (2.5 g, crude).

¹H-NMR: (400 MHz, D₂O): δ 8.84 (s, 1H), 4.05 (d, 1H), 3.98-3.95 (m, 1H),3.67 (s, 1H), 3.58 (d, 1H), 1.15 (d, 3H), 1.12 (d, 3H).

LCMS (ESI): 144.1 [M⁺+1]

Synthesis of 1-(pyrimidin-2-yl) propan-1-one (6S-AK)

To a stirring solution of pyrimidine-2-carbonitrile (20 g, 190 mmol) inTHF (200 mL) was added ethyl magnesium bromide (1M in THF, 227 mL, 228mmol) at 0° C. for 1 h. After completion of starting material (by TLC),the reaction mixture was diluted with saturated ammonium chloridesolution and EtOAc (150 mL). The separated organic layer was washed withbrine solution (2×100 mL). The extracted organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toafford crude material which was purified by column chromatographyeluting 20% EtOAc/hexane to afford 6S-AK (15 g, 57%) as an off-whitesolid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.00 (d, J=5.2 Hz, 2H), 7.70 (t, J=4.8 Hz,1H), 3.20-3.15 (m, 2H), 1.09 (t, J=7.2 Hz, 3H);

LCMS m/z: 137 [M⁺+1]

Synthesis of (Z)-2-(1-((triethylsilyl) oxy) prop-1-en-1-yl) pyrimidine(6S-AL)

To a stirring solution of 6S-AK (15 g, 110 mmol) in THF (100 mL) wasadded LiHMDS (1M in THF, 220 mL, 220 mmol) slowly at 0° C. and stirredfor 30 min. After added chloro triethylsilane (24.8 g, 165 mmol) in THF(50 mL) dropwise at 0° C. and stirred 1 h. After completion of startingmaterial (by TLC), the reaction mixture was diluted with saturatedammonium chloride solution (50 mL) and EtOAc (150 mL). The separatedorganic layer was extracted with brine solution (2×100 mL). Theseparated organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford crude material which waspurified by column chromatography eluting 5% EtOAc/hexane to afford6S-AI, (20 g, 74%) as yellow thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.75 (d, J=4.8 Hz, 2H), 7.32 (t, J=4.8 Hz,1H), 6.36-6.31 (m, 1H), 1.77 (d, J=7.2 Hz, 3H), 0.95-0.87 (m, 9H),0.71-0.65 (m, 6H).

Synthesis of 2-bromo-1-(pyrimidin-2-yl) propan-1-one (6S-AM)

To a stirring solution of 6S-AL (20 g, 80 mmol) in THF/H₂O (160 mL/40mL) were added N-bromosuccinimide (10.2 g, 88 mmol) slowly at RT andstirred for 2 h. After completion of starting material (by TLC), thereaction mixture was diluted with H₂O and EtOAc (100 ml/150 mL). Theseparated organic layer was washed with brine solution (2×100 mL). Theseparated organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford crude material which waspurified by column chromatography eluting 30% EtOAc/hexane to afford6S-AM (15 g, 87%) as yellow thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.06 (d, J=4.8 Hz, 2H), 7.75 (t, J=4.8 Hz,1H), 5.97-5.92 (m, 1H), 1.83 (d, J=6.4 Hz, 3H).

Synthesis of 2-hydroxy-1-(pyrimidin-2-yl) propan-1-one (6S-AN)

To a stirring solution of 6S-AM (15 g, 69 mmol) in MeOH (240 mL) wasadded sodium formate (18.9 g, 279 mmol) and stirred the reaction mass at70° C. for 8 h. After completion of reaction (by TLC), the reactionmixture was evaporated under reduced pressure to give crude product,which was purified by column chromatography eluting 5% MeOH/DCM toafford 6S-AN (5.5 g, 51%) as colorless liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.73 (d, J=5.2 Hz, 2H), 7.55 (t, J=4.8 Hz,1H), 5.28-5.26 (m, 1H), 1.24 (d, J=6.4 Hz, 1H), 0.99 (d, J=6.4 Hz, 3H)

Synthesis of (2-((tert-butyldimethylsilyl) oxy)-1-(pyrimidin-2-yl)propan-1-one (6S-AO)

To a stirring solution of 6S-AN (5.5 g, 36 mmol) in DCM (150 mL) wereadded imidazole (4.9 g, 72 mmol), DMAP (880 mg, 0.72 mmol) at 0° C. andstirred for 10 min. After added TBDMS-Cl (8.1 g, 54 mmol) at 0° C. andstirred at RT for 6 h. After completion of starting material (by TLC),diluted the reaction mass with H₂O (50 ml). The separated organic layerwas washed with brine solution (2×50 mL). The organic layer was driedover anhydrous Na₂SO₄, filtered and concentrated under reduced pressureto afford crude material which was purified by column chromatographyeluting 30% EtOAc/hexane to afford 6S-AO (3 g, 31%) as an off-whitesolid.

¹H-NMR: (400 MHz, CDCl₃): δ 9.00 (d, J=5.2 Hz, 2H), 7.71 (t, J=4.8 Hz,1H), 5.47-5.42 (m, 1H), 1.35 (d, J=6.8 Hz, 3H), 0.79 (s, 9H), 0.05 (s,6H)

Synthesis of (2-((tert-butyldimethylsilyl) oxy)-1-(pyrimidin-2-yl)propan-1-amine (6S-AP)

To a stirring solution of 6S-AO (3 g, 11.2 mmol) in MeOH (50 mL) wereadded sodium acetate (1.8 g, 22.5 mmol), ammonium carbonate (8.8 g, 56.3mmol), AcOH (0.6 mL, 11.2 mmol) at RT. The reaction mixture was thenstirred at 70° C. for 2 h. The temperature of the reaction was cooled toRT and added sodium cyanoborohydride (1.39 g, 22.5 mmol) and stirred at70° C. for 6 h. After consumption of starting material (by TLC), theMeOH was evaporated and the crude residue was diluted with DCM/H₂O (50ml/50 mL). The separated organic layer was washed with brine (2×50 mL).The organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford crude material waspurified by column chromatography eluting 5% MeOH/DCM to afford 6S-AP(2.4 g, 80%) as semi solid.

¹H-NMR: (400 MHz, CDCl₃): δ 8.83 (d, J=4.8 Hz, 2H), 7.40 (t, J=5.2 Hz,1H), 4.13 (t, J=6.4 Hz, 2H), 3.90 (d, J=6.4 Hz, 2H), 1.12 (d, J=6.4 Hz,3H), 0.70 (s, 9H), 0.02 (s, 6H).

Synthesis of 1-methyl-1H-1, 2, 4-triazole-5-carboxylic acid (6S-AQ)

To a stirred solution of 1-methyl-1H-1, 2, 4-triazole (2.0 g, 24.0 mmol)in THF (20 mL) was added n-butyl lithium (19 mL, 12.0 mmol) at −78° C.dropwise and stirred for 2 h. Solid CO₂ (2 g) was the added and thereaction stirred at −78° C. for 1 h. The reaction mass was stirred at RTfor 16 h. After consumption of the starting material (by TLC), thereaction was quenched with water (3 mL) and the obtained solid wasfiltered. The solid was triturated with diethyl ether/n-pentane (10mL/10 mL). The white color solid was dried on vacuum to afford 6S-AQ(2.0 g, 65.7%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.70 (s, 1H), 4.01 (s, 3H);

LCMS m/z: 128.3 [M⁺+1]

Synthesis of (Azidomethyl) Benzene (6S-AR)

To a stirring solution of benzyl bromide (30 g, 175 mmol) in dimethylformamide (300 mL) was added sodium azide (45.6 g, 701 mmol) at RT underinert atmosphere. The resultant reaction mixture was stirred at 70° C.for 16 h. After completion of reaction monitored (by TLC), the reactionmixture was allowed to RT; the volatiles were diluted with water (300mL) and ether (200 mL). The separated organic layer was washed by (3×200mL) of chilled water. After the separated organic layer was dried overanhydrous Na₂SO₄ filtered and concentrated under reduced pressure toafford 6S-AR (18 g, crude) as an off-white solid.

¹H-NMR: (400 MHz, CDCl₃): δ 7.40-7.29 (m, 5H), 4.32 (s, 2H).

Synthesis of Ethyl but-2-ynoate (6S-AS)

To a stirring solution of but-2-ynoic acid (2 g, 23.8 mmol) in dimethylformamide (20 mL) was added potassium carbonate (8.21 g, 59.5 mmol)slowly at 0° C. under inert atmosphere. After added ethyl iodide (2.85mL, 35.7 mmol) dropwise and the resultant reaction mixture was stirredat RT for 12 h. After completion of reaction monitored (by TLC), thereaction mixture was diluted with water (50 mL) and EtOAc (100 mL). Theseparated organic layer was washed by (3×100 mL) of chilled water andorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure to afford 6S-AS (1.4 g, crude) as reddish syrup.

¹H-NMR: (500 MHz, CDCl₃): δ 4.23 (q, J=6.5 Hz, 2H), 2.06 (s, 3H), 1.32(t, J=7.0 Hz, 3H);

Synthesis of ethyl 1-benzyl-5-methyl-1H-1, 2, 3-triazole-4-carboxylate(6S-AT-1 & 6S-AT-2)

To a stirring solution of 6S-AS (8.0 g, 71.3 mmol) in toluene (80 mL)was added 6S-AR (12.0 g, 107 mmol) at RT under inert atmosphere. Theresultant reaction mixture was heated to 100° C. and stirred for 16 h.The reaction mixture was allowed to RT; the volatiles were evaporatedunder reduced pressure to which, crude residue was purified by columnchromatography by eluting 40% EtOAc/hexane to afford two isomers 6S-AT-1(8 g) & 6S-AT-2 (8.2 g).

¹H-NMR (6S-AH-1): (400 MHz, CDCl₃): δ 7.30-7.26 (m, 5H), 5.86 (s, 2H),4.34 (q, J=7.2 Hz, 2H), 2.53 (s, 3H), 1.33 (t, J=7.2 Hz, 3H), LCMS m/z:246.3 [M⁺+1].

¹H-NMR (6S-AH-2): (400 MHz, CDCl₃): δ 7.36-7.31 (m, 3H), 7.16 (t, J=6.0Hz, 2H), 5.53 (s, 2H), 4.43 (q, J=7.2 Hz, 2H), 2.45 (s, 3H), 1.41 (t,J=7.2 Hz, 3H).

LCMS m/z: 246.3 [M⁺+1].

Synthesis of 1-benzyl-5-methyl-1H-1, 2, 3-triazole-4-carboxylic acid(6S-AU)

To a stirring solution of compound 6S-AT-2 (8.2 g, 33.4 mmol) in THF/H₂O(82 mL/82 mL, 1:1) was added LiOH.H₂O (4.2 g, 0.40 mmol) at RT andstirred for 16 h. After completion of reaction (by TLC), the volatileswere evaporated under reduced pressure. The residue was acidified withaqueous 2N HCl and the precipitated solid was filtered and washed withwater (25 mL), dried under reduced pressure to afford 6S-AU (7.0 g,97.2%) as an off-white solid.

¹H-NMR(H₂): (400 MHz, DMSO-d₆): δ 13.01 (br s, 1H), 7.40-7.32 (m, 5H),5.63 (s, 2H), 2.45 (s, 3H).

LCMS m/z: 218.3[M⁺+1]

Synthesis of 1-(azidomethyl)-3-fluorobenzene (6S-AV)

To a stirring solution of 1-(bromomethyl)-3-fluorobenzene (1 g, 5.29mmol) in dimethyl formamide (10 mL) was added sodium azide (859 mg, 13.2mmol) at RT under inert atmosphere. The resultant reaction mixture wasstirred at 90° C. for 12 h. After completion of reaction monitored (byTLC), the reaction mixture was allowed to RT; the volatiles were dilutedwith water (100 mL) and ethyl acetate (100 mL). The separated organiclayer was washed by (3×100 mL) of chilled water. After the separatedorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure to afford 6S-AV (700 mg, 87.7%) as yellow syrup.

¹H-NMR: (500 MHz, CDCl₃): δ 7.35-7.31 (m, 2H), 7.18-7.10 (m, 1H), 7.08(s, 1H), 4.40 (s, 2H)

IR: 2102 cm⁻¹

Synthesis of ethyl1-(3-fluorobenzyl)-5-methyl-1H-1,2,3-triazole-4-carboxylate (6S-AW-1 &6S-AW-2)

To a stirring solution of 6S-AV (2.4 g, 15.8 mmol) in toluene (10 mL)was added 6S-AG (1.78 g, 15.8 mmol) at RT under inert atmosphere. Theresultant reaction mixture was heated to 80° C. and stirred for 12 h.The reaction mixture was allowed to RT; the volatiles were evaporatedunder reduced pressure to which, crude residue was purified by columnchromatography by eluting 20% EtOAc/hexane to afford 6S-AW-1 (1 g, 24%)and 6S-AW-2 (1.5 g, 36.1%) as an off-white solid.

¹H-NMR (6S-AW-2, confirmed by NOE): (500 MHz, DMSO-d₆): δ 7.44-7.39 (m,1H), 7.19-7.15 (m, 1H), 7.03 (dd, J=10.5 Hz, 8.0 Hz, 2H), 5.67 (s, 2H),4.29 (q, J=7.5 Hz, 2H), 2.50 (s, 3H), 1.30 (t, J=7.0 Hz, 3H);

Mass m/z: 264.2 [M⁺+1]

Synthesis of 1-(3-fluorobenzyl)-5-methyl-1H-1, 2,3-triazole-4-carboxylic acid (6S-AX)

To a stirring solution of 6S-AW-2 (1.5 g, 5.7 mmol) in THF/H₂O (10 mL/5mL, 1:1), EtOH (2 mL) were added LiOH.H₂O (490 mg, 11.4 mmol) at RT andstirred for 6 h. After completion of reaction (by TLC), the volatileswere evaporated under reduced pressure. The residue was acidified withaqueous 2NHCl. The precipitated solid was filtered and co-distilled withtoluene (20 mL×2), dried under reduced pressure to afford 6S-AX (1.34 g,82%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.5 (br s, 1H), 4.16 (d, J=7.5 Hz, 2H),2.50 (s, 3H), 1.84-1.79 (m, 1H), 1.68-1.49 (m, 5H), 1.21-0.96 (m, 5H);

Mass m/z: 236.2[M⁺+1]

Synthesis of 1-(azidomethyl)-2-fluorobenzene (6S-AY)

To a stirring solution of 1-(bromomethyl)-2-fluorobenzene (1 g, 5.29mmol) in dimethyl formamide (10 mL) was added sodium azide (859 mg,13.22 mmol) at RT under inert atmosphere. The resultant reaction mixturewas stirred at 90° C. for 16 h. After completion of reaction monitored(by TLC), the reaction mixture was diluted with water (100 mL) and EtOAc(100 mL). The separated organic layer was washed by (3×100 mL) ofchilled water and organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to afford 6S-AY (700mg, crude) as yellow syrup.

¹H-NMR: (500 MHz, CDCl₃): δ 7.35-7.31 (m, 2H), 7.18-7.08 (m, 2H), 4.40(s, 2H);

IR: 2102 (cm⁻¹)

Synthesis of ethyl 1-(2-fluorobenzyl)-5-methyl-1H-1, 2,3-triazole-4-carboxylate (6S-AZ-1 & 6S-AZ-2)

To a stirring solution of 6S-AY (1.5 g, 13.39 mmol) in toluene (15 mL)was added 6S-AS (3.03 g, 20.08 mmol) at RT under inert atmosphere. Theresultant reaction mixture was heated to 100° C. and stirred for 16 h.After completion of the reaction, allowed to RT and the volatiles wereevaporated under reduced pressure to afford crude residue was purifiedby column chromatography by eluting 15% EtOAc/hexane to afford and6S-AZ-1 (500 mg, 14.2%) and 6S-AZ-2 (800 mg, 22.8%) as yellow solid.

¹H-NMR (6S-AZ-2, confirmed by NOESY1D): (500 MHz, CDCl₃): δ 7.33-7.30(m, 1H), 7.12-7.05 (m, 3H), 5.58 (s, 2H), 4.42 (q, J=7.0 Hz, 2H), 2.51(s, 3H), 1.41 (t, J=7.2 Hz, 3H);

Synthesis of 1-(2-fluorobenzyl)-5-methyl-1H-1, 2,3-triazole-4-carboxylic acid (6S-BA)

To a stirring solution of 6S-AZ-2 (800 mg, 3.04 mmol) in THF/H₂O (10mL/10 mL, 1:1) were added LiOH.H₂O (318 mg, 7.60 mmol) at RT and stirredfor 16 h. After completion of reaction (by TLC), the volatiles wereevaporated under reduced pressure. The residue was acidified withaqueous 2N HCl and the precipitated solid was filtered and washed withwater (5 mL) followed by n-pentane (5 mL) dried under reduced pressureto afford 6S-BA (600 mg, 84%) as an off-white solid.

¹H-NMR (I₂): (400 MHz, DMSO-d₆): δ 13.03 (br s, 1H), 7.45-7.39 (m, 1H),7.28-7.16 (m, 3H), 5.65 (s, 2H), 2.50 (s, 3H);

Mass m/z: 236.1[M⁺+1]

Synthesis of (Azidomethyl) Cyclohexane (6S-BB)

To a stirring solution of (bromomethyl) cyclohexane (2 g, 11.2 mmol) indimethylformamide (10 mL) was added sodium azide (2.18 g, 33.8 mmol) atRT under inert atmosphere. The resultant reaction mixture was stirred at90° C. for 12 h. After completion of reaction monitored (by TLC), thereaction mixture was allowed to RT; the volatiles were diluted withwater (100 mL) and ethyl acetate (100 mL). The separated organic layerwas washed by (3×100 mL) of chilled water. After the separated organiclayer was dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure to afford 6S-BB (1.0 g, 64.5%) as brown syrup.

¹H-NMR: (500 MHz, CDCl₃): δ 3.10 (t, J=7.0 Hz, 2H), 2.15-1.98 (m, 4H),1.77-1.66 (m, 4H), 1.56-1.53 (m, 1H), 1.00-0.95 (m, 2H)

Synthesis of ethyl 1-(cyclohexylmethyl)-5-methyl-1H-1, 2,3-triazole-4-carboxylate (6S-BC-1 & 6S-BC-2)

To a stirring solution of 6S-BB (1.0 g, 7.18 mmol) in toluene (10 mL)was added 6S-AS (1.20 g, 10.7 mmol) at RT under inert atmosphere. Theresultant reaction mixture was heated to 100° C. and stirred for 12 h.The reaction mixture was allowed to RT; the volatiles were evaporatedunder reduced pressure and the crude residue was purified by columnchromatography by eluting with 20% EtOAc/hexane to afford and 6S-BC-1(400 mg, 22.1%) and 6S-BC-2 (700 mg, 38.8%) as an off-white solid.

¹H-NMR (6S-BC-2, confirmed by Noesy): (400 MHz, CDCl₃): δ 4.42 (q, J=7.2Hz, 2H), 4.10 (d, J=7.6 Hz, 2H), 2.56 (s, 3H), 1.95-1.88 (m, 1H),1.76-1.69 (m, 3H), 1.68-1.61 (m, 2H), 1.41-1.33 (m, 3H), 1.03 (t, J=11.6Hz, 3H), 0.97-0.89 (m, 2H);

Mass m/z: 252.3 [M⁺+1]

Synthesis of 1-(cyclohexylmethyl)-5-methyl-1H-1, 2,3-triazole-4-carboxylic acid (6S-BD)

To a stirring solution of 6S-BC-2 (700 mg, 2.78 mmol) in THF/H₂O (10mL/5 mL, 1:1), EtOH (1 mL) were added LiOH.H₂O (230 mg, 5.57 mmol) at RTand stirred for 6 h. After completion of reaction (by TLC), thevolatiles were evaporated under reduced pressure. The residue wasacidified with aqueous 2N HCl. The precipitated solid was filtered andwashed with water (2 mL), dried under reduced pressure to afford 6S-BD(600 mg, 96.7%) as an off-white solid.

¹H-NMR (6S-BD): (500 MHz, DMSO-d₅): δ 12.5 (br s, 1H), 4.16 (d, J=7.5Hz, 2H), 2.50 (s, 3H), 1.84-1.79 (m, 1H), 1.68-1.49 (m, 5H), 1.21-0.96(m, 5H);

Mass m/z: 224.2[M⁺+1]

Synthesis of 1-(cyclohexylmethyl)-5-methyl-1H-1, 2,3-triazole-4-carbonyl chloride (6S-BE)

To a stirring solution of 6S-BD (200 mg, 0.89 mmol) in CH₂Cl₂ (5 mL),DMF (0.1 mL) were added oxalyl chloride (0.14 mL, 17.8 mmol) at 0° C.The reaction mixture was warmed to RT and stirred for 3 h. Afterformation of the acid chloride volatiles were evaporated under reducedpressure in presence of N₂ atmosphere to afford 6S-BE (210 mg, crude).

Synthesis of Cyclopentylmethyl Methanesulfonate (6S-BF)

To a stirring solution of cyclopentylmethanol (2 g, 20 mmol) in CH₂Cl₂(20 mL) was added Et₃N (7.21 mL, 50 mmol) and mesyl chloride (2.31 mL,30 mmol) at −78° C. and stirred at RT for 2 h. After completion ofstarting material (by TLC), the organic layer was washed with water(1×30 mL) followed by brine solution (1×50 mL). After the separatedorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure to afford 6S-BF (2 g, crude) as brown syrup. Theobtained crude was used for next step without any further purification.

¹H-NMR: (500 MHz, CDCl₃): δ4.11 (d, J=7.5 Hz, 2H), 3.00 (s, 3H),2.33-2.27 (m, 1H), 1.83-1.77 (m, 2H), 1.65-1.57 (m, 4H), 1.32-1.25 (m,2H)

Synthesis of (Azidomethyl) Cyclopentane (6S-BG)

To a stirring solution of 6S-BF (2 g (crude), 11.2 mmol) in DMF (20 mL)was added sodium azide (2.19 g, 33.7 mmol) at RT and heated to 80° C.for 16 h. After completion of starting material (by TLC), the reactionmass was diluted with water (100 mL) and EtOAc (50 mL). The separatedorganic layer was washed with water (1×100 mL) followed by brinesolution (1×100 mL). After the separated organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toafford 6S-BG (1.2 g, crude) as brown syrup. The obtained crude was usedfor next step without any further purification.

¹H-NMR: (400 MHz, CDCl₃): δ3.20 (d, J=7.2 Hz, 2H), 2.18-2.11 (m, 1H),1.84-1.76 (m, 2H), 1.69-1.53 (m, 4H), 1.28-1.20 (m, 2H)

Synthesis of ethyl 1-(cyclopentylmethyl)-5-methyl-1H-1, 2,3-triazole-4-carboxylate (6S-BH-1 & 6S-BH-2)

To a stirring solution of 6S-BG (1.2 g (crude), 9.6 mmol) in toluene (15mL) was added 6S-AS (1.29 g, 11.5 mmol) at RT and heated to 100° C. for16 h. After completion of starting material (by TLC), evaporatedreaction mass under reduced pressure to obtain crude which was purifiedby column chromatography by eluting 10% EtOAc/n-hexane to afford 6S-BH-1(300 mg, 13.2%) and 6S-BH-2 (600 mg, 26.4%) as white solid.

¹H-NMR: (6S-BH-2, identified by NOESY): (400 MHz, CDCl₃): δ4.45-4.39 (m,2H), 4.21 (d, J=8.0 Hz, 2H), 2.57 (s, 3H), 2.46-2.42 (m, 1H), 1.73-1.56(m, 6H), 1.43 (d, J=7.2 Hz, 3H), 1.34-1.25 (m, 2H)

LCMS m/z: 238.2 [M⁺+1]

1-(cyclopentylmethyl)-5-methyl-1H-1, 2, 3-triazole-4-carboxylic acid(6S-BI)

To a stirred solution of 6S-BH-2 (600 mg, 2.53 mmol) in THF: H₂O (10mL/3 mL) were added LiOH.H₂O (265 mg, 6.30 mmol) at RT and stirred for16 h. After consumption of the starting material (by TLC), the volatileswere evaporated under reduced pressure. The residue was diluted withwater (10 mL) and acidified to pH˜2 using citric acid. The obtainedprecipitate was filtered and triturated with n-hexane (10 mL) to afford6S-BI (400 mg, 75.7%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.9 (br s, 1H), 4.24 (d, J=7.6 Hz, 2H),2.51 (s, 3H), 2.40-2.32 (m, 1H), 1.62-1.50 (m, 6H), 1.27-1.21 (m, 2H);

LCMS m/z: 210.2 [M⁺+1]

Synthesis of 1-(azidomethyl)-4-fluorobenzene (6S-BJ)

To a stirring solution of 1-(bromomethyl)-4-fluorobenzene (1 g, 5.29mmol) in dimethylformamide (10 mL) was added sodium azide (859 mg, 13.22mmol) at RT under inert atmosphere. The resultant reaction mixture wasstirred at 80° C. for 12 h. After completion of reaction monitored (byTLC), the reaction mixture was diluted with water (100 mL) and EtOAc(100 mL). The separated organic layer was washed by (3×100 mL) ofchilled water and organic layer was dried over anhydrous Na₂SO₄ filteredand concentrated under reduced pressure to afford 6S-BJ (800 mg, crude)as yellow syrup.

IR: (2100 cm⁻¹)

Synthesis of ethyl 1-(4-fluorobenzyl)-5-methyl-1H-1, 2,3-triazole-4-carboxylate (6S-BK-1& 6S-BK-2)

To a stirring solution of 6S-AS (500 mg, 4.46 mmol) in toluene (5 mL)was added 6S-BJ (1.01 g, 6.69 mmol) at RT under inert atmosphere. Theresultant reaction mixture was heated to 100° C. and stirred for 12 h.After completion of the reaction, allowed to RT and the volatiles wereevaporated under reduced pressure to afford crude residue was purifiedby column chromatography by eluting 30% EtOAc/hexane to afford 6S-BK-1(250 mg, 21.3%) and 6S-BK-2 (400 mg, 34.1%) as yellow solid.

¹H-NMR (6S-BK-2, confirmed by NOESY1D): (500 MHz, CDCl₃): δ 7.18-7.15(m, 2H), 7.04 (t, 0.1=8.5 Hz, 2H), 5.50 (s, 2H), 4.41 (q, 0.1=7.5 Hz,2H), 2.46 (s, 3H), 1.41 (t, J=7.0 Hz, 3H);

Synthesis of 1-(4-fluorobenzyl)-5-methyl-1H-1, 2,3-triazole-4-carboxylic acid (6S-BL)

To a stirring solution of 6S-BK-2 (800 mg, 3.04 mmol) in THF/H₂O (5 mL/5mL, 1:1) were added LiOH.H₂O (318 mg, 7.60 mmol) at RT and stirred for16 h. After completion of reaction (by TLC), the volatiles wereevaporated under reduced pressure. The residue was acidified withaqueous 2N HCl and the precipitated solid was filtered and washed withwater (5 mL) followed by n-pentane (5 mL) dried under reduced pressureto afford 6S-BL (400 mg, 56%) as white solid.

¹H-NMR (6S-BL): (500 MHz, DMSO-d₆): δ 13.02 (br s, 1H), 7.28 (t, J=8.0Hz, 2H), 7.20 (t, J=9.0 Hz, 2H), 5.61 (s, 2H), 2.51 (s, 3H);

Mass m/z: 236.1[M⁺+1]

Synthesis of 1-(azidomethyl)-4-methoxybenzene (6S-BM)

To a stirring solution of 1-(chloromethyl)-4-methoxybenzene (2 g, 12.7mmol) in dimethyl formamide (20 mL) was added sodium azide (2.07 g, 31.7mmol) at RT under inert atmosphere. The resultant reaction mixture wasstirred at 100° C. for 12 h. After completion of reaction as indicatedby TLC, the reaction mixture was allowed to cool to RT; the volatileswere diluted with water (200 mL) and ethyl acetate (200 mL). Theseparated organic layer was washed by (3×200 mL) of chilled water andorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure to afford 6S-BM (1.4 g, crude) as reddish syrup.

¹H-NMR: (500 MHz, CDCl₃): δ 7.24 (d, J=8.5 Hz, 2H), 6.91 (d, J=8.5 Hz,2H), 4.26 (s, 2H), 3.81 (s, 3H);

IR: 2097 cm⁻¹

Synthesis of 4-(ethoxymethyl)-1-(4-methoxybenzyl)-5-methyl-1H-1, 2,3-triazole (6S-BN-1 & 6S-BN-2)

To a stirring solution of 6S-AS (1 g, 8.92 mmol) in toluene (10 mL) wasadded 6S-BM (2.18 g, 13.39 mmol) at RT under inert atmosphere. Theresultant reaction mixture was heated to 100° C. and stirred for 12 h.The reaction mixture was allowed to cool to RT; the volatiles wereevaporated under reduced pressure to which, crude residue was purifiedby column chromatography by eluting 20% EtOAc/hexane to afford 6S-BN-1(200 mg, 8.1%) and 6S-BN-2 (300 mg, 12.2%)

¹H-NMR (6S-BN-2, confirmed by NOESY): (500 MHz, CDCl₃): δ 7.12 (d, J=8.5Hz, 2H), 6.86 (d, J=8.5 Hz, 2H), 5.46 (s, 2H), 4.41 (q, J=7.5 Hz, 2H),3.78 (s, 3H), 2.45 (s, 3H), 1.41 (t, J=7.0 Hz, 3H)

Synthesis of 1-(4-methoxybenzyl)-5-methyl-1H-1, 2,3-triazole-4-carboxylic acid (6S-BO)

To a stirring solution of 6S-BN-2 (300 mg, 1.09 mmol) in THF/H₂O (2 mL/2mL, 1:1) were added LiOH.H₂O (114 mg, 2.72 mmol) at RT and stirred for 6h. After completion of reaction (by TLC), the volatiles were evaporatedunder reduced pressure. The residue was acidified with aqueous 2N HCland the precipitated solid was filtered. The obtained solid wastriturated with n-pentane (2 mL), dried under reduced pressure to afford6S-BO (130 mg, 48%) as a white solid.

¹H-NMR (6S-BO): (500 MHz, DMSO-d₆): δ 12.85 (br s, 1H), 7.17 (d, J=8.5Hz, 2H), 6.91 (d, J=8.0 Hz, 2H), 5.53 (s, 2H), 3.72 (s, 3H), 2.50 (s,3H)

Synthesis of 1-(azidomethyl)-4-methylbenzene (6S-BP)

To a stirring solution of 1-(bromomethyl)-4-methylbenzene (1 g, 5.40mmol) in dimethyl formamide (15 mL) was added sodium azide (878 mg,13.51 mmol) at RT under inert atmosphere. The resultant reaction mixturewas stirred at 100° C. for 12 h. After completion of reaction monitored(by TLC), the reaction mixture was allowed to RT; the volatiles werediluted with water (100 mL) and ethyl acetate (2×100 mL). The separatedorganic layer was washed by (3×200 mL) of chilled water and the organiclayer was dried over anhydrous Na₂SO₄ filtered and concentrated underreduced pressure to afford 6S-BP (700 mg, crude) as brown syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.26-7.20 (m, 4H), 4.37 (s, 2H), 2.30 (s,3H);

IR: 2097 cm⁻¹

Synthesis of ethyl 5-methyl-1-(4-methylbenzyl)-1H-1, 2,3-triazole-4-carboxylate (6S-BO-1 & 6S-BQ-2)

To a stirring solution of 6S-AS (4.72 g, 42.5 mmol) in toluene (30 mL)was added 6S-BP (2.5 g, 17.0 mmol) at RT under inert atmosphere. Theresultant reaction mixture was heated to 100° C. and stirred for 12 h.The reaction mixture was allowed to RT; the volatiles were evaporatedunder reduced pressure to which, crude residue was purified by columnchromatography by eluting 50% EtOAc/hexane to afford 6S-BQ-1 (1.5 g,33.7%) and 6S-BQ-2 (2 g, 45%) as a yellow sticky solid.

¹H-NMR (6S-BQ-2, confirmed by NOESY): (500 MHz, CDCl₃): δ 7.14 (d, J=7.5Hz, 2H), 7.05 (d, J=8.0 Hz, 2H), 5.48 (s, 2H), 4.40 (q, J=7.5 Hz, 2H),2.44 (s, 3H), 2.32 (s, 3H), 1.40 (t, J=7.0 Hz, 3H)

Synthesis of 5-methyl-1-(4-methylbenzyl)-1H-1, 2,3-triazole-4-carboxylic acid (6S-BR)

To a stirring solution of 6S-BQ-2 (500 mg, 1.92 mmol) in THF/H₂O (10mL/10 mL, 1:1) were added LiOH.H₂O (201 mg, 4.80 mmol) at RT and stirredfor 6 h. After completion of reaction (by TLC), the volatiles wereevaporated under reduced pressure. The residue was acidified withaqueous 2N HCl (pH˜2) and the precipitated solid was filtered. Theobtained solid was triturated with n-pentane (2 mL), dried under reducedpressure to afford 6S-BR (350 mg, 78.8%) as a white solid.

¹H-NMR (6S-BR): (500 MHz, CDCl₃): δ 7.16 (d, J=8.0 Hz, 2H), 7.08 (d,J=8.0 Hz, 2H), 5.49 (s, 2H), 2.48 (s, 3H), 2.33 (s, 3H);

LCMS m/z: 232.1 [M⁺+1]

Synthesis of 5-methyl-1-(4-methylbenzyl)-1H-1, 2, 3-triazole-4-carbonylchloride (6S-BS)

To a stirring solution of 6S-BR (250 mg, 1.08 mmol) in CH₂Cl₂ (10 mL),DMF (0.1 mL) were added oxalyl chloride (0.18 mL, 2.16 mmol) at 0° C.The reaction mixture was warmed to RT and stirred for 2 h. The volatileswere evaporated under reduced pressure in presence of N₂ atmosphere toafford acid chloride 6S-BS (250 mg, crude).

Synthesis of ethyl2-(5-acetyl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetate (6S-BT)

To a stirring solution of 6S-L (12.5 g, 36 mmol) in EtOAc (100 mL) wereadded acetic anhydride (7.36 g, 72.2 mmol), 50% wet 10% Pd/C (5.0 g) andstirred under H₂ atmosphere (balloon pressure) for 4 h at RT. Aftercompletion of reaction (by TLC), the reaction mixture was filteredthrough a pad of celite and triturated with EtOAc (50 mL). The filtratewas concentrated under reduced pressure to afford 6S-BT (8.0 g, 87.9%)as yellow syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ4.21 (s, 1H), 4.17 (s, 1H), 4.14-4.12 (m,1H), 3.82 (s, 1H), 3.68 (d, J=4.8 Hz, 1H), 3.56-3.51 (m, 1H), 3.46-3.43(m, 1H), 3.29 (d, J=4.8 Hz, 2H), 2.11-2.09 (m, 1H), 1.97 (s, 2H),1.90-1.89 (m, 3H), 1.20 (t, J=7.2 Hz, 3H).

2-(5-acetyl-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl) acetic acid (6S-BU)

To a stirred solution of 6S-BT (8.0 g, 31.49 mmol) in THF: H₂O (80 mL/30mL) were added LiOH.H₂O (3.30 g, 78.7 mmol) at RT and stirred for 2 h.After consumption of the starting material (by TLC), the volatiles wereevaporated under reduced pressure. The residue was diluted with water(25 mL), extracted with ether (2×50 mL). The separated aqueous layer wasacidified to pH˜2 using 2N HCl and extracted with 5% MeOH/DCM (3×50 mL).The organic layers were dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to afford 6S-BU (6.5 g, 91.5%) as an off-whitesolid.

¹H-NMR: (500 MHz, DMSO-d₆): δ12.5 (br s, 1H), 4.12 (s, 1H), 3.70 (s,1H), 3.66-3.64 (m, 2H), 3.53-3.51 (m, 2H), 3.53-3.51 (m, 2H), 3.42 (d,J=7.0 Hz, 1H), 2.73-2.63 (m, 2H), 2.10-2.07 (m, 1H);

LCMS m/z: 227.2 [M⁺+1]

Synthesis of ethyl 2-(1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetate(6S-BV)

To a stirring solution of 6S-L (3.8 g, 10.98 mmol) in methanol (50 mL)was added 50% wet 10% Pd/C (800 mg) and stirred under H₂ atmosphere(balloon pressure) for 2 h at RT. The reaction mixture was filteredthrough a pad of celite and triturated with methanol (50 mL). Thefiltrate was concentrated under reduced pressure to afford 6S-BV (2 g,86.2%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ1.11-3.92 (m, 4H), 3.40-3.26 (m, 4H),1.94-1.91 (m, 2H), 1.75-1.67 (m, 2H), 1.91 (t, J=7.5 Hz, 3H);

Synthesis of ethyl2-(5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetate (6S-BW)

To a stirring solution of 6S-BV (2.0 g, 9.43 mmol) in CH₂Cl₂ (20 mL) wasadded TEA (3.25 mL, 23.58 mmol) followed by isobutyryl chloride (1.07mL, 14.14 mmol) at 0° C. and stirred for 3 h at RT. After the reactionwas completed, the reaction mass was diluted with water (30 mL). Theseparated organic layer was washed by brine solution (1×50 mL). Theorganic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to afford 6S-BW (2.4 g, crude) as thick syrup. Thismaterial was directly used for the next step without furtherpurification.

¹H-NMR: (500 MHz, DMSO-d₆): δ 4.20 (s, 2H), 4.14-4.08 (m, 1H), 3.80-3.76(m, 1H), 3.62-3.57 (m, 2H), 3.51-3.46 (m, 2H), 2.68-2.63 (m, 1H),2.09-2.05 (m, 2H), 1.89-1.85 (m, 2H), 1.21-1.16 (m, 3H), 1.03-0.95 (m,6H);

Synthesis of 2-(5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)aceticacid (6S-BX)

To a stirred solution of 6S-BW (2.4 g (crude), 8.51 mmol) in THF: H₂O(20 mL/20 mL) were added LiOH.H₂O (893 mg, 21.27 mmol) at RT and stirredfor 4 h. After consumption of the starting material (by TLC), thevolatiles were evaporated under reduced pressure. The residue wasdiluted with water (20 mL) and washed with EtOAc (1×50 mL). Theseparated aqueous layer was acidified to pH˜4 using citric acid andextracted with EtOAc (2×50 mL). The organic layers were washed withbrine, dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to afford 6S-BX (1.2 g, 57%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.83 (br s, 1H), 4.13 (s, 2H), 3.70-3.46(m, 4H), 2.70-2.64 (m, 1H), 2.10-2.06 (m, 2H), 1.91-1.85 (m, 2H),1.01-0.96 (m, 6H)

Synthesis of benzyl 2-(2-(((2R, 3S)-3-acetoxy-1-amino-1-oxobutan-2-yl)amino)-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate(6S-FNL-2)

To a stirred solution of 6S-M (0.1 g, 0.31 mmol) in DMF (2 mL) was addedHATU (141 mg, 0.37 mmol) followed by 6S-D (59 mg, 0.37 mmol) and DIPEA(0.14 mL, 0.37 mmol). The resultant reaction mixture was allowed to warmto RT and stirred for 10 h. It was quenched with water and extractedwith EtOAc (2×20 mL). The organic layer was washed with brine, driedover Na₂SO₄ and concentrated under reduced pressure. Purification bycolumn chromatography afforded (6S-FNL-2) (30 mg, 21%).

¹H-NMR: (400 MHz, CDCl₃): δ 7.38-7.27 (m, 5H), 7.06 (d, J=8.0 Hz, 1H),6.99 (s, 2H), 5.55-5.49 (m, 1H), 5.43-5.39 (m, 1H), 5.34 (s, 2H),4.79-4.76 (m, 1H), 4.39-4.11 (m, 1H), 3.94-3.89 (m, 2H), 3.84-3.70 (m,1H), 3.62-3.53 (m, 1H), 2.41-2.36 (m, 1H), 2.23-2.15 (m, 1H), 2.08 (s,3H), 2.02-1.88 (m, 2H), 1.35-1.22 (m, 3H)

Synthesis of (2R, 3S)-4-amino-4-oxo-3-(2-(1-oxo-2, 5-diazaspiro [3.4]octan-2-yl) acetamido) butan-2-yl acetate (6S-FNL-3)

To a stirred solution of (6S-FNL-2) (0.4 g, 0.86 mmol) in MeOH (20 mL)was added 10% Pd/C (0.1 g) under inert atmosphere. The resultingreaction mixture was agitated under H₂ atmosphere (balloon pressure) for4 h at RT. The reaction mixture was filtered through a celite pad andthe filtrate was concentrated under reduced pressure. The crude wastriturated with ether to afford (6S-FNL-3) (0.2 g, 70%).

¹H-NMR: (500 MHz, DMSO-d₆): δ8.04 (s, 1H), 7.54 (br s, 1H), 7.18 (s,2H), 5.18-5.12 (m, 1H), 4.48-4.42 (m, 1H), 3.98-3.92 (m, 2H), 3.41-3.37(m, 1H), 3.29-3.26 (m, 1H), 2.92-2.87 (m, 2H), 1.98-1.87 (m, 5H),1.77-1.72 (m, 2H), 1.18-1.12 (m, 3H).

Synthesis of Benzyl 2-(2-((1-amino-3-hydroxy-1-oxobutan-2-yl)amino)-2-oxoethyl)-1-oxo-2,5-diazaspiro [3.4] octane-5-carboxylate(6S-FNL-4)

To a stirring solution of 6S-M (0.1 g, 0.31 mmol) in DMF (2 mL) wereadded 6S-F (55 mg, 0.47 mmol), DIPEA (96.87 mg, 0.78 mmol), HATU (143mg, 0.37 mmol) at 0° C. and under inert atmosphere. The resultantreaction mixture was allowed to warm to RT and stirred for 16 h. Thereaction mixture was quenched with ice cold water and extracted withEtOAc (2×20 mL). The organic layer was washed with water followed bybrine, dried over anhydrous Na₂SO₄ and concentrated under reducedpressure. Purification by column chromatography affords (6S-FNL-4) (40mg, 30%).

¹H-NMR: (400 MHz, CDCl₃): δ 8.09-7.36 (m, 1H), 7.35-7.33 (m, 5H), 6.60(br s, 1H), 5.37 (br s, 1H), 5.09 (s, 2H), 4.56-4.39 (m, 3H), 4.13-3.99(m, 1H), 3.70-3.51 (m, 4H), 3.48-3.39 (m, 1H), 2.43-2.39 (m, 1H),2.27-2.21 (m, 1H), 2.07-1.91 (m, 1H), 1.59-1.42 (m, 1H), 1.28-1.20 (m,3H)

Synthesis of (2S,3R)-3-hydroxy-2-(2-(1-oxo-2,5-diazaspiro[3.4]octan-2-yl) acetamido)butanamide (6S-FNL-5)

To a stirring solution of (6S-FNL-4) (0.2 g, 0.47 mmol) in MeOH (10 mL)was added 10% Pd/C (40 mg) under inert atmosphere. The resultingreaction mixture was agitated under H₂ atmosphere (balloon pressure) for4 h at RT. The reaction mixture was filtered through a celite pad andthe filtrate was concentrated under reduced pressure. The crude wastriturated with ether to afford (6S-FNL-5) (0.11 g, 83%).

¹H-NMR: (400 MHz, CD₃OD): δ 4.23-4.21 (m, 1H), 4.20-4.10 (m, 1H), 4.05(s, 2H), 3.53-3.47 (m, 2H), 3.12-3.00 (m, 2H), 2.22-2.07 (m, 2H),2.01-1.82 (m, 2H), 1.18 (d, J=6.4 Hz, 3H)

UPLC (ELSD purity): 99.9%

Synthesis of tert-butyl ((2S, 3R)-1-(2-(2-(((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)amino)-2-oxoethyl)-1-oxo-2,5-diazaspiro [3.4] octan-5-yl)-3-hydroxy-1-oxobutan-2-yl) carbamate(6S-1)

To a stirring solution of (6S-FNL-5) (300 mg, 1.05 mmol) in DCM (25 mL),DMF (0.5 mL) were added N, N-diisopropylethylamine (0.58 mL, 3.15 mmol),6S-U (277 mg, 1.26 mmol), followed by HATU (481 mg, 1.26 mmol) at 0° C.and stirred at RT for 16 h. After consumption of the starting material(by TLC), the reaction mixture was evaporated under reduced pressure andthe obtained crude was purified by column chromatography by eluting 6%MeOH/DCM to afford crude (400 mg, 70% pure by LCMS), further purified bypreparative chromatography to yield pure 6S-1 (230 mg, 44.9% yield) asan off-white solid.

Preparative column: Kromasil 250×21.2, 10 μm

Mobile phase: n-Hexane: [DCM:MeOH (80:20)]

¹H-NMR: (500 MHz, DMSO-d₆): δ7.83 (d, J=8.5 Hz, 1H), 7.25 (s, 2H), 6.33(d, J=9.0 Hz, 1H), 4.80-4.75 (m, 2H), 4.24-3.98 (m, 4H), 3.71-3.60 (m,3H), 3.39-3.33 (m, 1H), 2.14-1.89 (m, 4H), 1.41 (s, 9H), 1.14-1.05 (m,6H);

LCMS m/z: 486.3 [M⁺+1]

Synthesis of (2S, 3R)-2-(2-(5-((2S,3R)-2-amino-3-hydroxybutanoyl)-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl)acetamido)-3-hydroxybutanamide (6S-FNL-6)

To a stirring solution of compound 6S-1 (130 mg, 0.26 mmol) in DCM (3mL) was added TFA (152 mg, 1.34 mmol) at 0° C. and stirred at RT for 2h. After completion of starting material (by TLC), the reaction mixturewas concentrated under reduced pressure and co-distilled with a mixtureof solvents pentane (2 mL), diethyl ether (2 mL), DCM (1 mL) to afford(6S-FNL-6) (110 mg, 82.7% LCMS purity 93.32%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ4.33-4.28 (m, 1H), 4.23-4.19 (m, 4H),4.05-4.01 (m, 1H), 3.91-3.87 (m, 1H), 3.71-3.65 (m, 2H), 3.55-3.49 (m,1H), 2.30-2.24 (m, 2H), 2.02-1.97 (m, 2H), 1.27 (t, J=4.0 Hz, 3H), 1.15(t, J=5.5 Hz, 3H);

LCMS m/z: 386.3 [M⁺+1]

(2S,3R)-2-(2-(5-acetyl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamido)-3-hydroxybutanamide (6S-FNL-7)

To a stirring solution of (6S-FNL-4) (1 g, 2.39 mmol) in EtOAc (50 mL)was added acetic anhydride (0.48 g, 4.78 mmol) followed by Pd/C (0.5 g)under N₂ atmosphere. The reaction mixture was stirred at RT for 16 hunder H₂ atmosphere. After consumption of the starting material (byTLC), the reaction mixture was filtered through a pad of celite andwashed with EtOAc (20 mL). Obtained filtrate was concentrated underreduced pressure to afford crude compound was purified by columnchromatography. The obtained mixture of compound was purified by chiralpreparative HPLC to afford (6S-FNL-7-F1) in fraction-I (0.075 g),(6S-FNL-7-F2) in fraction-II (0.062 g) as off-white solids.

¹H-NMR (Fr-1): (400 MHz, DMSO-d₆): δ7.87 (d, J=8.0 Hz, 1H), 7.24 (s,1H), 7.02 (s, 1H), 4.84 (d, J=7.2 Hz, 1H), 4.10-4.03 (m, 2H), 3.96 (s,2H), 3.79 (d, J=5.2 Hz, 1H), 3.61-3.56 (m, 1H), 3.51-3.45 (m, 2H),2.19-2.15 (m, 2H), 2.00 (s, 3H), 1.92-1.87 (m, 2H), 1.05 (d, J=6.4 Hz,3H).

LCMS m/z: 327.3 [M⁺+1].

HPLC Purity: Fr-I (91.20%), Fr-II (97.03%)

¹H-NMR (Fr-II): (400 MHz, DMSO-d₆): δ7.85 (d, J=8.4 Hz, 1H), 7.17 (s,1H), 7.07 (s, 1H), 4.79 (d, J=6.8 Hz, 1H), 4.09-4.06 (m, 2H), 3.97 (s,2H), 3.70 (d, J=4.8 Hz, 1H), 3.60-3.55 (m, 1H), 3.51-3.45 (m, 2H),2.18-2.13 (m, 2H), 1.99 (s, 3H), 1.92-1.85 (m, 2H), 1.05 (d, J=6.4 Hz,3H);

LCMS m/z: 327.4 [M⁺+1]

HPLC Purity: Fr-II 97.03%

(2S,3R)-3-hydroxy-2-(2-(5-(1-methyl-1H-1,2,4-triazole-5-carbonyl)-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamido)butanamide (6S-FNL-8)

To a stirring solution of (NRX-2079) (6S-FNL-5) (0.25 g, 0.88 mmol) inCH₂Cl₂ (20 mL) was added HOBt (178 mg, 1.32 mmol), EDCI.HCl (0.2 g, 1.00mmol) followed by DIPEA (0.4 mL, 2.20 mmol) and 6S-AQ (134 mg, 1.05mmol) at 0° C. The reaction mixture was stirred at RT for 16 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasconcentrated under reduced pressure to obtain crude product. Thismaterial was purified by column chromatography followed by prep-HPLCpurification to afford NRX-2310 (6S-FNL-8) (0.07 g, 21%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.09 (s, 1H), 7.89 (t, 1H), 7.25 (d, 1H),7.12 (t, 1H), 4.93 (s, 1H), 4.19-4.15 (m, 2H), 4.03 (s, 3H), 3.96-3.91(m, 4H), 3.44 (d, 1H), 2.25-2.20 (m, 3H), 1.97-1.91 (m, 2H), 1.07 (s,3H).

LCMS (m/z): 394.2[M⁺+1]

HPLC Purity: 93%

Benzyl 2-(2-(((2S, 3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl) butan-2-yl)amino)-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate(6S-2)

To a stirring solution of 6S-M (1 g, 3.14 mmol) in CH₂Cl₂ (50 mL) wasadded EDCI (719 mg, 3.76 mmol), HOBt (635 mg, 4.71 mmol) followed byDTPEA (2.8 mL, 15.7 mmol) and 6S-W (784 mg, 3.77 mmol) at 0° C. Thereaction mixture was stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction was quenched with water andextracted with CH₂Cl₂ (2×50 mL). The organic layer was dried overanhydrous Na₂Sa₄ and concentrated under reduced pressure. The crude waspurified by column chromatography to afford 6S-2 (0.8 g, 54%).

LCMS (m/z): 473.4 [M⁺+1]

N-((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-2-(1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamide(6S-3)

To a stirring solution of 6S-2 (0.8 g, 1.69 mmol) in CH₃OH (60 mL) wasadded Pd/C (0.4 g) under N₂ atmosphere. The reaction mixture was stirredat RT for 4 h under H₂ atmosphere. After consumption of the startingmaterial (by TLC), the reaction mixture was filtered through a pad ofcelite and washed with MeOH. Obtained filtrate was concentrated underreduced pressure to afford 6S-3 (0.5 g) as crude. This material wasdirectly used for the next step without further purification.

LCMS (m/z): 339.3 [M⁺+1]

N-((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-2-(5-(1-methyl-1H-1,2,4-triazole-5-carbonyl)-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamide(6S-FNL-9)

To a stirring solution of 6S-3 (0.315 g, 2.48 mmol) in CH₂Cl₂ (60 mL)was added EDCI.HCl (336 mg, 1.76 mmol), HOBt (297 mg, 2.20 mmol), DIPEA(0.67 mL, 3.67 mmol) and 6S-AQ (0.5 g, 1.47 mmol) at 0° C. The reactionmixture was stirred at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with DCM and washedwith water. The separated organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to obtain crudeproduct. This material was purified by column chromatography followed byprep-HPLC purification to afford (6S-FNL-9): (80 mg, 12%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.09 (s, 1H), 4.55-4.51 (m, 1H), 4.08 (d,2H), 3.97 (s, 2H), 3.87-3.84 (m, 3H), 3.70-3.55 (m, 2H), 3.45 (t, 1H),3.35-3.31 (m, 2H), 2.75 (s, 3H), 2.27-2.24 (m, 2H), 1.98-1.92 (m, 4H),1.85-1.84 (m, 2H), 1.19 (d, 3H).

LCMS (m/z): 448[M⁺+1]

HPLC Purity: 94%

Synthesis of2-(5-acetyl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)-N-((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)acetamide(6S-FNL-10)

To a stirring solution of 6S-2 (300 mg, 0.63 mmol) in EtOAc (20 mL) wasadded acetic anhydride (0.13 g, 1.27 mmol) followed by Pd/C (150 mg)under N₂ atmosphere. The reaction mixture was stirred at RT for 16 hunder H₂ atmosphere. After consumption of the starting material (byTLC), the reaction mixture was filtered through a pad of celite andwashed with EtOAc (20 mL). Obtained filtrate was concentrated underreduced pressure to afford crude compound was purified by columnchromatography by eluting with 2% MeOH/DCM to afford (6S-FNL-10) (90 mg,37.5%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ8.08 (t, J=8.4 Hz, 1H), 4.83-4.75 (m, 1H),4.44-4.38 (m, 1H), 3.93-3.83 (m, 3H), 3.72-3.67 (m, 1H), 3.64-3.57 (m,3H), 3.55-3.46 (m, 1H), 3.29-3.23 (m, 3H), 2.13-2.09 (m, 2H), 2.07 (s,3H), 1.98-1.82 (m, 4H), 1.79-1.73 (m, 2H), 1.11-0.98 (m, 3H);

LCMS m/z: 381.3 [M⁺+1];

HPLC Purity: 94.2%

Synthesis of benzyl 2-(2-(((1S, 2R)-2-hydroxy-1-(1, 3, 4-oxadiazol-2-yl)propyl)amino)-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4]octane-5-carboxylate (6S-4)

To a stirring solution of 6S-M (600 mg, 4.19 mmol) in DCM (20 mL) wereadded N, N-diisopropylethylamine (1.93 mL, 10.4 mmol), 6S-AD (1.60 g,5.02 mmol), followed by HATU (1.91 g, 5.02 mmol) at 0° C. and stirred atRT for 16 h. After consumption of the starting material (by TLC), thereaction mixture was diluted with water (20 mL). The separated organiclayer was washed with saturated NaHCO₃ solution (1×30 mL) followed bybrine solution (1×20 mL). The separated organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to afford 6S-4(600 mg, 33.3%) as pale yellow syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.21 (s, 1H), 7.35-7.32 (m, 5H), 5.13 (d,J=4.5 Hz, 1H), 5.13 (s, 2H), 5.09-5.00 (m, 1H), 4.12-3.94 (m, 2H),3.70-3.33 (m, 6H), 2.16-2.11 (m, 4H), 1.08 (d, J=6.5 Hz, 3H);

LCMS m/z: 444.5 [M⁺+1]

Synthesis of N-((1S, 2R)-2-hydroxy-1-(1, 3, 4-oxadiazol-2-yl)propyl)-2-(1-oxo-2, 5-diazaspiro [3.4] octan-2-yl) acetamide (6S-FNL-11)

To a stirring solution of 6S-4 (600 mg, 1.35 mmol) in MeOH (10 mL) wereadded (50% wet) 10% Pd/C (200 mg) and stirred under H₂ atmosphere(balloon pressure) for 3 h at RT. After completion of reaction (by TLC),the reaction mixture was filtered through a pad of celite and trituratedwith EtOAc/MeOH (10 mL/10 mL). The filtrate was concentrated underreduced pressure to afford (6S-FNL-11) (400 mg, crude) as yellow syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.21 (s, 1H), 8.68 (s, 1H), 5.29-5.07 (m,1H), 4.10-3.91 (m, 2H), 3.61 (d, J=16.5 Hz, 1H), 3.40-3.31 (m, 1H),3.16-2.93 (m, 2H), 2.02-1.91 (m, 2H), 1.80-1.76 (m, 2H), 1.32-1.28 (m,1H), 1.24 (d, J=6.5 Hz, 1H), 1.09 (d, J=6.5 Hz, 3H);

LCMS m/z: 310.2 [M⁺+1];

Synthesis of N-((1S, 2R)-2-hydroxy-1-(1, 3, 4-oxadiazol-2-yl)propyl)-2-(5-(1-methyl-1H-1, 2, 4-triazole-5-carbonyl)-1-oxo-2,5-diazaspiro [3.4] octan-2-yl) acetamide (6S-FNL-12)

To a stirring solution of (6S-FNL-11) (300 mg, 0.97 mmol) in DCM (30 mL)were added N, N-diisopropylethylamine (0.44 mL, 2.42 mmol), 6S-AQ (147mg, 1.16 mmol), followed by HATU (442 mg, 1.10 mmol) at 0° C. andstirred at RT for 16 h. After consumption of the starting material (byTLC), the reaction mixture was concentrated under reduced pressure togive crude product, which was purified by column chromatography by 4%MeOH/DCM to afford yellow syrup was purified by preparative HPLC methodpurification to afford (6S-FNL-12) (80 mg, 19.7%) as colorless thicksyrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.15 (s, 1H), 8.52 (t, J=8.0 Hz, 1H), 8.08(s, 1H), 5.19-5.16 (m, 1H), 4.04 (s, 2H), 4.02 (s, 3H), 3.94-3.84 (m,5H), 3.44-3.41 (m, 1H), 2.27-2.21 (m, 2H), 1.93-1.85 (m, 2H), 1.10-1.08(m, 3H);

LCMS m/z: 419 [M⁺+1];

HPLC: 96.64%

Synthesis ofN-((1S,2R)-2-hydroxy-1-(1,3,4-oxadiazol-2-yl)propyl)-2-(5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamide(6S-FNL-13)

To a stirred solution of (6S-FNL-11) (250 mg, 0.8 mmol) in DCM (20 mL)was added TEA (242 mg, 2.4 mmol) at 0° C. After added isobutyrylchloride (102 mg, 0.96 mmol) slowly and the resulting reaction mixturewas stirred at RT for 3 h. After consumption of the starting material(by TLC), the reaction mixture was diluted with water (10 mL). Theorganic layer was washed by citric acid (1×20 mL) followed by brinesolution (1×20 mL). The separated organic layer was dried over anhydrousNa₂SO₄ and concentrated under reduced pressure to obtain crude product,which was purified by silica gel column chromatography eluting with 2%MeOH/CH₂Cl₂ followed by preparative HPLC purification to afford compound(6S-FNL-13) (80 mg, 26.3%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ9.19 (s, 1H), 8.64-8.57 (m, 1H), 5.17-5.13(m, 1H), 5.12-5.08 (m, 1H), 4.17-4.09 (m, 2H), 3.84-3.76 (m, 1H),3.70-3.64 (m, 2H), 3.57-3.51 (m, 1H), 3.37-3.34 (m, 1H), 2.77-2.70 (m,1H), 2.17-2.05 (m, 2H), 1.90-1.79 (m, 2H), 1.13 (d, J=6.0 Hz, 3H),1.03-0.97 (m, 6H);

LCMS m/z: 380.4 [M⁺+1];

HPLC: 98.5% (both enantiomers)

2-(5-acetyl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)-N-((1S,2R)-2-hydroxy-1-(1,3,4-oxadiazol-2-yl)propyl)acetamide (6S-FNL-14)

To a stirring solution of 6S-BU (200 mg, 1.39 mmol) in DCM (10 mL) wereadded N,N-diisopropylethylamine (0.64 mL, 3.47 mmol), 6S-AD (5.95 g,37.7 mmol), followed by HATU (637 mg, 1.68 mmol) at 0° C. and stirred atRT for 16 h. After consumption of the starting material (by TLC), thereaction mixture was concentrated under reduced pressure to give crudeproduct, which was purified by column chromatography by 8% MeOH/DCM toafford yellow syrup was purified by preparative HPLC method purificationto afford (6S-FNL-14) (100 mg, 20.4%) as colorless liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ9.18 (d, J=3.6 Hz, 1H), 8.54 (t, J=7.6 Hz,1H), 5.15-5.06 (m, 2H), 4.16-4.12 (m, 1H), 4.05-4.00 (m, 1H), 3.89-3.82(m, 1H), 3.72 (t, J=5.6 Hz, 1H), 3.61-3.56 (m, 1H), 3.51-3.45 (m, 1H),3.37-3.33 (m, 1H), 2.18-2.08 (m, 2H), 2.02 (s, 3H), 1.93-1.90 (m, 2H),1.10 (t, J=6.4 Hz, 3H)

LCMS m/z: 352.3 [M⁺+1];

UPLC: 47.2%&44.3%

Synthesis of tert-butyl2-(2-(((1S,2R)-2-hydroxy-1-(1,3,4-oxadiazol-2-yl)propyl)amino)-2-oxoethyl)-1-oxo-2,5-diazaspiro[3.4]octane-5-carboxylate(6S-FNL-15)

To a stirring solution of 6S-T (500 mg, 1.76 mmol) in DCM (20 mL) wereadded DIPEA (0.88 mL, 5.1 mmol), 6S-AD (302 mg, 2.11 mmol) followed byHATU (801 mg, 2.11 mmol) at 0° C. and stirred for 12 h at RT. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with water (10 mL). The separated organic layer was washed withsaturated NaHCO₃ solution (1×20 mL) followed by brine solution (1×20mL). The organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. Obtained crude material waspurified by silica gel column chromatography eluting with 2% MeOH/DCM toafford (6S-FNL-15) (220 mg, 31.6%) as sticky syrup.

¹H-NMR: (400 MHz, D₂O): δ8.99 (s, 1H), 5.46-5.38 (m, 1H), 4.80 (s, 2H),4.56-4.43 (m, 1H), 4.14-3.93 (m, 1H), 3.64-3.46 (m, 3H), 2.38-2.34 (m,2H), 2.00-1.96 (m, 2H), 1.50 (s, 9H), 1.34 (d, J=6.0 Hz, 3H);

LCMS (m/z): 410.6 [M⁺+1];

HPLC: 92.4%

2-(5-Acetyl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)-N-((1S,2R)-2-hydroxy-1-(1,2,4-oxadiazol-5-yl)propyl)acetamide(6S-FNL-16)

To a stirring solution of 6S-BU (0.5 g, 2.21 mmol) in CH₂Cl₂ (20 mL) wasadded EDCI (0.63 g, 3.31 mmol), HOBT (0.44 g, 3.31 mmol) and 6S-AJ (0.37g, 2.65 mmol) followed by DIPEA (1.4 g, 10.85 mmol) at 0° C. Thereaction mixture was stirred at RT for 12 h. After consumption of thestarting material (by TLC), the reaction was diluted with water andextracted with CH₂Cl₂ (2×20 mL). The organic layer was dried overanhydrous Na₂SO₄ and concentrated under vacuum. The crude was purifiedby column chromatography by eluting 2% MeOH: DCM to afford (6S-FNL-16)(0.09 g, 12%) as yellow liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ8.94 (s, 1H), 8.61 (t, J=8.4 Hz, 1H),5.23-5.20 (m, 1H), 5.16-5.09 (m, 1H), 4.20-4.01 (m, 1H), 3.91-3.85 (m,1H), 3.73-3.71 (m, 1H), 3.65 (s, 1H), 3.59-3.57 (m, 1H), 3.49-3.34 (m,1H), 2.16-2.09 (m, 2H), 2.05 (s, 3H), 1.98-1.89 (m, 3H), 1.13-1.11 (m,3H).

LCMS m/z: 352.2 [M⁺+1]

Synthesis ofN-((1S,2R)-2-hydroxy-1-(1,2,4-oxadiazol-5-yl)propyl)-2-(5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamide(6S-FNL-17)

To a stirring solution of 6S-BX (500 mg, 1.96 mmol) in DMF (5 mL) wereadded N, N-diisopropylethylamine (1.15 mL, 6.86 mmol), 6S-AJ (604 mg,2.35 mmol) followed by HATU (893 mg, 2.35 mmol) at 0° C. and stirred atRT for 12 h. After consumption of the starting material (by TLC), thereaction mixture was diluted with water (50 mL) and EtOAc (50 mL). Theseparated organic layer was washed with citric acid solution (1×100 mL),brine solution (2×50 mL) followed by water (2×50 mL). The separatedorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure to give crude product, which was purified bycolumn chromatography by 2% MeOH/DCM to afford (6S-FNL-17) (128 mg,17.2%) as sticky solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ8.94 (s, 1H), 8.68-8.60 (m, 1H), 5.22-5.10(m, 2H), 4.20-4.07 (m, 2H), 3.86-3.79 (m, 1H), 3.71-3.65 (m, 2H),3.58-3.51 (m, 1H), 3.38-3.34 (m, 1H), 2.77-2.66 (m, 1H), 2.15-1.91 (m,4H), 1.14 (d, J=4.0 Hz, 3H), 1.03-0.97 (m, 6H);

LCMS m/z: 380.4 [M⁺+1];

UPLC: 94.01%

Synthesis of tert-butyl2-(2-((1S,2R)-2-hydroxy-1-(1,2,4-oxadiazol-5-yl)propyl)amino)-2-oxoethyl)-1-oxo-2,5-diazaspiro[3.4]octane-5-carboxylate(6S-FNL-18)

To a stirring solution of 6S-T (1 g, 3.51 mmol) in DMF (10 mL) wereadded DIPEA (1.75 mL, 10.53 mmol), EDCI (1.0 g, 5.26 mmol), HOBT (710mg, 5.26 mmol) followed by 6S-AJ (602 mg, 4.21 mmol) at 0° C. andstirred for 16 h at RT. After consumption of the starting material (byTLC), the reaction mixture was diluted with water (15 mL). The separatedorganic layer was washed with brine solution (1×20 mL). The organiclayer was dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure. Obtained crude material was purified by silica gelcolumn chromatography eluting with 2% MeOH/DCM to afford (6S-FNL-18)(150 mg, 10.5%) as thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ8.95 (s, 1H), 8.52 (d, J=7.6 Hz, 1H), 5.25(d, J=4.8 Hz, 1H), 5.19-5.10 (m, 1H), 4.21-4.17 (m, 2H), 3.86-3.80 (m,1H), 3.71-3.67 (m, 1H), 3.55 (t, J=4.4 Hz, 2H), 3.27-3.23 (m, 1H),2.13-2.08 (m, 2H), 1.82-1.74 (m, 2H), 1.39 (s, 9H), 1.10 (d, J=6.4 Hz,3H);

Mass (ESI): m/z 432.4 [M⁺+Na];

HPLC: 98.35% (both isomers)

2-(5-acetyl-1-oxo-2, 5-diazaspiro [3.4]octan-2-yl)-N-(2-((tert-butyldimethylsilyl) oxy)-1-(Pyrimidin-2-yl)propyl) acetamide (6S-5)

To a stirring solution of 6S-BU (400 mg, 1.77 mmol) in DCM (20 mL) wereadded N, N-diisopropylethylamine (0.9 mL, 5.32 mmol), 6S-AP (474 mg,1.77 mmol), EDCI.HCl (509 mg, 2.66 mmol), HOBT (410 mg, 2.66 mmol) at 0°C. and stirred at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was concentrated under reducedpressure to give crude product, which was purified by columnchromatography by 4% MeOH/DCM to afford 6S-5 (300 mg, 35%) as colorlessthick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.78 (d, J=16.0 Hz, 2H), 8.35 (d, J=9.0Hz, 1H), 7.37 (d, =16.0 Hz, 1H), 4.88 (t, =8.0 Hz, 1H), 4.37 (d, =8.0Hz, 1H), 3.90 (d, J=8.0 Hz, 2H), 3.85 (s, 2H), 3.76 (d, J=10.0 Hz, 1H),3.63-3.57 (m, 1H), 3.49-3.43 (m, 1H), 3.13 (d, J=9.0 Hz, 1H), 2.13-2.10(m, 2H), 2.01 (s, 3H), 1.16 (d, J=5.5 Hz, 3H), 0.64 (s, 9H), 0.06 (s,6H); LCMS m/z: 474.6 [M⁺−1]

Synthesis of 2-(5-acetyl-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl)-N-((1R,2R)-2-hydroxy-1-(pyrimidin-2-yl) propyl) acetamide (6S-FNL-19)

To a stirring solution of 6S-5 (300 mg, 0.63 mmol) in THF (20 mL) wasadded TBAF (1.26 mL) slowly at 0° C. and stirred at RT for 2 h. Aftercompletion of reaction (by TLC), the reaction mixture was evaporated togive crude product, which was purified by column chromatography eluting4% MeOH/DCM to afford mixture (110 mg) of isomers again purified bychiral preparative HPLC method purification to afford (6S-FNL-19) (60mg, 26%) as colorless liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.76 (t, J=6.0 Hz, 2H), 8.28 (d, J=8.8 Hz,1H), 7.37 (t, J=4.8 Hz, 1H), 4.91-4.79 (m, 2H), 4.15-4.10 (m, 1H),3.92-3.78 (m, 2H), 3.67 (d, J=4.8 Hz, 1H), 3.60-3.54 (m, 1H), 3.49-3.31(m, 2H), 2.13-2.11 (m, 2H), 2.09 (s, 3H), 2.08-1.87 (m, 2H), 1.09 (d,J=6.4 Hz, 3H);

LCMS m/z: 362.4 [M⁺+1];

HPLC: 97.5%

Synthesis of tert-butyl 2-(2-((2-((tert-butyldimethylsilyl)oxy)-1-(pyrimidin-2-yl) propyl) amino)-2-oxoethyl)-1-oxo-2, 5-diazaspiro[3.4] octane-5-carboxylate (6S-6)

To a stirring solution of 6S-T (300 mg, 1.05 mmol) in DCM (20 mL) wereadded DIPEA (0.55 mL, 3.16 mmol), 6S-AP (338 mg, 1.26 mmol) followed byBOP (696 mg, 1.57 mmol) at 0° C. and stirred for 12 h at RT. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with water (15 mL). The separated organic layer was washed withbrine solution (1×20 mL). The organic layer was dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure. Obtained crudematerial was purified by silica gel column chromatography eluting with2% MeOH/DCM to afford 6S-6 (150 mg, 26%) as sticky syrup.

LCMS (m/z): 534.7 [M⁺+1]

Synthesis of tert-butyl 2-(2-((2-hydroxy-1-(pyrimidin-2-yl) propyl)amino)-2-oxoethyl)-1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate(6S-FNL-20)

To a stirring solution of 6S-6 (300 mg, 0.56 mmol) in THF (10 mL) wasadded TBAF (1M in THF) (1.11 mL, 1.12 mmol) at 0° C. under N₂ atmosphereand stirred at RT for 2 h. After consumption of the starting material(by TLC), the reaction mixture was concentrated under reduced pressureto obtain crude residue which was diluted with water (10 mL) and EtOAc(20 mL). The separated organic layer was washed with brine solution(1×30 mL). The organic layer was dried over anhydrous Na₂SO₄, filteredand concentrated under reduced pressure to afford crude compound whichwas purified by preparative HPLC purification to obtained (6S-FNL-20)(40 mg, 17%) as white solid.

¹H-NMR: (400 MHz, CD₃OD): 8.79-8.72 (m, 2H), 7.39-7.34 (m, 1H),5.13-5.08 (m, 1H), 4.81-4.70 (m, 1H), 4.50-4.40 (m, 1H), 4.35-4.21 (m,1H), 3.91-3.75 (m, 2H), 3.48-3.37 (m, 3H), 2.33-2.22 (m, 2H), 1.97-1.85(m, 2H), 1.41 (s, 9H), 1.18-1.16 (m, 3H);

LCMS (m/z): 420.5 [M⁺+1];

HPLC: 99.3%

Synthesis of benzyl2-(2-((2-((tert-butyldimethylsilyl)oxy)-1-(pyrimidin-2-yl)propyl)amino)-2-oxoethyl)-1-oxo-2,5-diazaspiro[3.4]octane-5-carboxylate(6S-7)

To a stirring solution of 6S-M (1.3 g, 4.08 mmol) in DCM (30 mL) wereadded N, N-diisopropylethylamine (2.1 mL, 12.2 mmol), 6S-AP (1.09 g,4.08 mmol) followed by EDCI.HCl (1.1 g, 6.13 mmol) HOBT (938 mg, 6.13mmol) at 0° C. and stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was diluted with water(30 mL). The separated organic layer was washed with brine solution(1×50 mL). The separated organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure to afford crude which waspurified by column chromatography by eluting 4% MeOH/DCM to obtained6S-7 (1.5 g, 65%) as yellow thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ8.80-8.74 (m, 2H), 7.39-7.34 (m, 6H), 5.09(s, 2H), 4.93 (t, J=4.8 Hz, 1H), 4.38-4.19 (m, 1H), 4.05-3.68 (m, 2H),3.49-3.39 (m, 4H), 2.20-2.11 (m, 2H), 1.86-1.85 (m, 2H), 1.19-1.10 (m,4H), 0.65 (s, 9H), −0.07 (s, 3H), −0.03 (s, 3H);

LCMS m/z: 568 [M⁺+1]

Synthesis ofN-(2-((tert-butyldimethylsilyl)oxy)-1-(pyrimidin-2-yl)propyl)-2-(1-oxo-2,5-diazaspiro [3.4] octan-2-yl)acetamide (6S-8)

To a stirring solution of 6S-7 (500 mg, 0.88 mmol) in EtOAc (25 mL) wasadded (50% wet) 10% Pd/C (250 mg) and stirred under H₂ atmosphere(balloon pressure) at RT for 7 h. After completion of reaction (by TLC),the reaction mixture was filtered through a pad of celite and trituratedwith EtOAc (10 mL). The filtrate was concentrated under reduced pressureto afford 6S-8 (320 mg, crude) as yellow thick syrup. This compound wasused directly for next step without any purification.

LCMS m/z: 434.5 [M⁺+1]

Synthesis ofN-(2-((tert-butyldimethylsilyl)oxy)-1-(pyrimidin-2-yl)propyl)-2-(5-(1-methyl-1H-1,2,4-triazole-5-carbonyl)-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamide(6S-9)

To a stirring solution of 6S-AQ (93 mg, 0.73 mmol) in DCM (20 mL) wereadded N, N-diisopropylethylamine (0.4 mL, 2.21 mmol), 6S-8 (320 mg, 0.73mmol), followed by EDCI.HCl (211 mg, 1.10 mmol), HOBT (170 mg, 1.10mmol) at 0° C. and stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was evaporated underreduced pressure and the obtained crude was purified by columnchromatography by eluting 4% MeOH/DCM to afford 6S-9 (210 mg, crude) asyellow thick syrup. This compound was used directly for next stepwithout any purification.

LCMS m/z: 543.5 [M⁺+1]

Synthesis ofN-(2-hydroxy-1-(pyrimidin-2-yl)propyl)-2-(5-(1-methyl-1H-1,2,4-triazole-5-carbonyl)-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamide(6S-FNL-21)

To a stirring solution of 6S-9 (210 mg crude, 0.38 mmol) in THF (5 mL)was added TBAF in THF (0.77 ml, 0.76 mmol) at 0° C. and stirred at RTfor 3 h. After consumption of the starting material (by TLC), thereaction mixture was evaporated under reduced pressure and the obtainedcrude was purified by column chromatography by eluting 4% MeOH/DCMfollowed by preparative TLC to afford (6S-FNL-21) (70 mg, 42%) as yellowthick syrup.

¹H-NMR: (400 MHz, CD₃OD): δ8.75-8.70 (m, 2H), 7.95 (s, 1H), 7.36-7.30(m, 1H), 5.16-5.13 (m, 1H), 4.53 (s, 1H), 4.41-4.43 (m, 2H), 4.29-4.03(m, 1H), 3.99 (s, 3H), 3.96-3.81 (m, 2H), 3.58-3.54 (m, 1H), 2.37-2.34(m, 2H), 2.09-2.00 (m, 2H), 1.22 (d, J=6.4 Hz, 3H);

LCMS m/z: 429 [M⁺+1].

Synthesis of(2S,3R)-3-hydroxy-2-(2-(5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamido)butanamide(6S-FNL-22)

To a stirring solution of 6S-BX (500 mg, 1.96 mmol) in DMF (3 mL) wereadded N, N-diisopropylethylamine (1.02 mL, 5.88 mmol), 6S-F (277 mg,2.35 mmol) followed by HATU (893 mg, 2.35 mmol) at 0° C. and stirred atRT for 16 h. After consumption of the starting material (by TLC), thereaction mixture was quenched with brine solution (15 mL) and extractedwith 10% MeOH/DCM (2×15 mL). The combined organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure togive crude product, which was purified by column chromatography by 2%MeOH/DCM to afford (6S-FNL-22) (100 mg, 14.4%) as white solid.

¹H-NMR: (400 MHz, D₂O): δ4.37-4.34 (m, 2H), 4.08-3.96 (m, 2H), 3.84-3.79(m, 1H), 3.72-3.68 (m, 1H), 3.67-3.61 (m, 1H), 2.93-2.86 (m, 1H),2.37-2.25 (m, 2H), 2.11-2.02 (m, 2H), 1.38-1.29 (m, 4H), 1.14-1.09 (m,6H);

LCMS m/z: 355.5 [M⁺+1];

HPLC: 99.97%

Synthesis of tert-butyl2-(2-(((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)amino)-2-oxoethyl)-1-oxo-2,5-diazaspiro[3.4]octane-5-carboxylate(6S-FNL-23)

To a stirring solution of 6S-T (2 g, 7.04 mmol) in DMF (10 mL) wereadded DIPEA (3.78 mL, 21.12 mmol), 6S-F (997 mg, 8.44 mmol) followed byHATU (3.2 g, 8.44 mmol) at 0° C. and stirred for 16 h at RT. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with water (100 mL) and extracted with EtOAc (2×100 mL). Theorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure. Obtained crude material was purified by silicagel column chromatography eluting with 2% MeOH/DCM to afford (6S-FNL-23)(1.1 g, 40.7%) as white solid.

¹H-NMR: (400 MHz, D₂O): δ4.33-4.26 (m, 3H), 4.21-4.12 (m, 1H), 4.05-3.86(m, 1H), 3.59-3.55 (m, 2H), 3.53-3.35 (m, 1H), 2.33-2.27 (m, 2H),1.97-1.90 (m, 2H), 1.46 (s, 9H), 1.25-1.22 (m, 3H);

Mass (ESI): in/z 383.4 [M⁺+1];

HPLC: 97.8% (both isomers)

Synthesis of (2R, 3S)-4-amino-3-(2-(5-(1-benzyl-5-methyl-1H-1, 2,3-triazole-4-carbonyl)-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl)acetamido)-4-oxobutan-2-yl acetate (6S-FNL-24)

To a stirring solution of 6S-AU (4.0 g, 18.43 mmol) in CH₂Cl₂ (20 mL),DMF (0.1 mL) were added oxalyl chloride (3.34 mL, 36.86 mmol) at 0° C.The reaction mixture was warmed to RT and stirred for 2 h. The volatileswere evaporated under reduced pressure in presence of N₂ atmosphere toafford acid chloride. To a stirred solution of acid chloride in DCM (40mL) was added (6S-FNL-3) (3.8 g, 11.65 mmol), N, N-diisopropylethylamine(6.44 mL, 37.04 mmol) at 0° C. The resulting reaction mixture wasstirred at RT for 2 h. After consumption of the starting material (byTLC), the reaction mixture was diluted with water (30 mL) and extractedwith CH₂Cl₂ (2×30 mL). Combined organic extracts were washed by NaHCO₃(2×25 mL). The separated organic extracts were dried over anhydrousNa₂SO₄ and concentrated under reduced pressure to give crude product,which was purified by silica gel column chromatography eluting with 2%MeOH/CH₂Cl₂ to afford compound (6S-FNL-24) (2.6 g, 42.5%) as anoff-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.87, 7.89 (dd, J=8.5 Hz, J=8.5 Hz, 1H),7.47 (d, J=11.0 Hz, 2H), 7.38-7.31 (m, 2H), 7.17 (t, 3H), 5.63 (s, 2H),5.20-5.16 (m, 1H), 4.43-4.41 (m, 1H), 4.24-4.14 (m, 1H), 4.02 (t, J=7.0Hz, 1H), 3.84 (s, 3H), 3.38-3.33 (m, 1H), 2.20-2.14 (m, 2H), 1.97 (s,3H), 1.92-1.80 (m, 5H), 1.16 (t, J=7.5 Hz, 3H)

LCMS m/z: 526.6 [M⁺+1];

HPLC: 51.34%&46.08% (enantiomers)

Synthesis of (2R, 3S)-4-amino-3-(2-(5-(1-(4-fluorobenzyl)-5-methyl-1H-1,2, 3-triazole-4-carbonyl)-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl)acetamido)-4-oxobutan-2-yl acetate (6S-FNL-25)

To a stirring solution of 6S-BL (100 mg, 0.42 mmol) in CH₂Cl₂ (5 mL),DMF (0.1 mL) were added oxalyl chloride (108 mg, 0.85 mmol) at 0° C. Thereaction mixture was warmed to RT and stirred for 2 h. The volatileswere evaporated under reduced pressure in presence of N₂ atmosphere toafford acid chloride (150 mg, crude). To a stirred solution of acidchloride (150 mg, 0.59 mmol, crude) in DCM (10 mL) were added (6S-FNL-3)(193 mg, 0.59 mmol), N. N-diisopropylethylamine (229 mg, 1.77 mmol) at0° C. The resulting reaction mixture was stirred at RT for 8 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with water (10 mL) and extracted with CH₂Cl₂ (2×20 mL). Combinedorganic extracts were washed by saturated NaHCO3 solution (2×30 mL)followed by citric acid solution (1×20 mL). The separated organicextracts were dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to obtain crude product, which was purified by silica gelcolumn chromatography eluting with 2% MeOH/CH₂Cl₂ to afford compound(6S-FNL-25) (100 mg, 31.2%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ7.99 (dd, J=8.4 Hz, 8.8 Hz, 1H), 7.48 (d,J=9.6 Hz, 1H), 7.28-7.16 (m, 5H), 5.63 (s, 2H), 5.20-5.16 (m, 1H),4.44-4.40 (m, 1H), 4.20 (d, J=7.6 Hz, 1H), 3.92-3.81 (m, 3H), 3.39-3.29(m, 2H), 2.50 (s, 3H), 2.22-2.15 (m, 2H), 1.92-1.86 (m, 2H), 1.81 (s,3H), 1.16 (d, J=6.4 Hz, 3H);

LCMS m/z: 544.6 [M⁺+1];

HPLC: 97.6% (both enantiomers)

Synthesis of(2R,3S)-4-amino-3-(2-(5-(1-(4-methoxybenzyl)-5-methyl-1H-1,2,3-triazole-4-carbonyl)-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamido)-4-oxobutan-2-ylacetate (6S-FNL-26)

To a stirring solution of 6S-BO (140 mg, 0.57 mmol) in CH₂Cl₂ (5 mL),DMF (0.2 mL) were added oxalyl chloride (0.1 mL, 1.14 mmol) at 0° C. Thereaction mixture was warmed to RT and stirred for 2 h. The volatileswere evaporated under reduced pressure in presence of N₂ atmosphere toafford acid chloride (160 mg, crude). To a stirred solution of acidchloride (160 mg, crude) in DCM (10 mL) was added (6S-FNL-3) (186 mg,0.57 mmol), diisopropylethylamine (220 mg, 1.71 mmol) at 0° C. Theresulting reaction mixture was stirred at RT for 1.5 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with water (50 mL) and extracted with CH₂Cl₂ (2×30 mL). Combinedorganic extracts were washed by brine solution (2×50 mL). The separatedorganic extracts were dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to obtain crude product, which was purified by silicagel column chromatography eluting with 5% MeOH/CH₂Cl₂ to afford compound(6S-FNL-26) (79 mg, 24%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ8.17 (t, J=9.5 Hz, 1H), 7.42 (d, J=18.8 Hz,1H), 7.15 (d, J 8.0 Hz, 2H), 6.91 (d, J=8.0 Hz, 2H), 5.55 (s, 2H),5.17-5.16 (m, 1H), 4.46-4.45 (m, 1H), 3.99-3.91 (m, 2H), 3.73 (s, 3H),3.40-3.36 (m, 1H), 2.63 (s, 3H), 2.40-2.36 (m, 2H), 2.21-2.14 (m, 2H),1.98 (s, 3H), 1.96-1.92 (m, 2H), 1.40 (s, 2H), 1.12 (d, J=6.0 Hz, 3H)

LCMS m/z: 556.6 [M⁺+1];

HPLC: 92.2% (both enantiomers)

Synthesis of (2R,3S)-4-amino-3-(2-(5-(5-methyl-1-(4-methylbenzyl)-1H-1,2,3-triazole-4-carbonyl)-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamido)-4-oxobutan-2-ylacetate (6S-FNL-27)

To a stirred solution of 6S-BS (250 mg, crude, 1 mmol) in DCM (10 mL)was added (6S-FNL-3) (391 mg, 1.2 mmol), N, N-diisopropylethylamine (387mg, 3 mmol) at 0° C. The resulting reaction mixture was stirred at RTfor 8 h. After consumption of the starting material (by TLC), thereaction mixture was diluted with water (20 mL) and extracted withCH₂Cl₂ (2×30 mL). Combined organic extracts were washed by NaHCO₃solution (2×20 mL) followed by citric acid solution (2×20 mL). Theseparated organic extracts were dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to obtain crude product, which waspurified by silica gel column chromatography eluting with 2% MeOH/CH₂Cl₂to afford compound (6S-FNL-27) (100 mg, 18.5%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ7.97 (dd, J=9.2 Hz, 9.6 Hz, 1H), 7.47 (d,J=8.8 Hz, 1H), 7.17 (d, J=8.0 Hz, 3H), 7.07 (d, J=8.0 Hz, 2H), 5.58 (s,2H), 5.20-5.14 (m, 1H), 4.44-4.40 (m, 1H), 4.25 (s, 2H), 3.92-3.80 (m,3H), 3.38 (dd, J=15.2 Hz, 14.8 Hz, 1H), 2.37 (s, 3H), 2.27 (s, 3H),2.20-2.15 (m, 2H), 1.92-1.86 (m, 2H), 1.81 (s, 3H), 1.16 (d, J=6.4 Hz,3H)

LCMS m/z: 540.6 [M⁺+1];

HPLC: 98.3% (both enantiomers)

Synthesis of(2R,3S)-4-amino-3-(2-(5-(1-(3-fluorobenzyl)-5-methyl-1H-1,2,3-triazole-4-carbonyl)-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamido)-4-oxobutan-2-ylacetate (6S-FNL-28)

To a stirred solution of 6S-AX (170 mg, 0.72 mmol) in DCM (10 mL) wereadded (6S-FNL-3) (235 mg, 0.72 mmol), N, N-diisopropylethylamine (280mg, 2.17 mmol), HATU (547 mg, 1.44 mmol) at 0° C. The resulting reactionmixture was stirred at RT for 18 h. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with water (10 mL)and extracted with CH₂Cl₂ (2×30 mL). Combined organic extracts werewashed by NaHCO₃ solution (1×20 mL), citric acid solution (1×20 mL)followed by brine solution (2×30 mL). The separated organic layers weredried over anhydrous Na₂SO₄ and concentrated under reduced pressure toobtain crude product, which was purified by silica gel columnchromatography eluting with 2% MeOH/CH₂Cl₂ to afford compound(6S-FNL-28) (185 mg, 47.3%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ7.95 (dd, J=8.8 Hz, 8.4 Hz, 1H), 7.50-7.39(m, 2H), 7.17 (t, J=11.2 Hz, 2H), 7.01 (dd, J=9.6 Hz, 7.6 Hz, 2H), 5.75(s, 2H), 5.21-5.16 (m, 1H), 4.44-4.40 (m, 1H), 4.26-4.15 (m, 1H),4.06-4.03 (m, 1H), 3.95-3.81 (m, 3H), 3.39-3.34 (m, 1H), 2.39 (s, 3H),2.23-2.12 (m, 2H), 1.98 (s, 3H), 1.93-1.81 (m, 2H), 1.17 (d, J=6.4 Hz,3H);

LCMS m/z: 544.3 [M⁺+1];

HPLC: 97.4% (both enantiomers)

Synthesis of(2R,3S)-4-amino-3-(2-(5-(1-(2-fluorobenzyl)-5-methyl-1H-1,2,3-triazole-4-carbonyl)-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamido)-4-oxobutan-2-ylacetate (6S-FNL-29)

To a stirring solution of 6S-BA (100 mg, 0.42 mmol) in CH₂Cl₂ (5 mL),DMF (0.1 mL) were added oxalyl chloride (108 mg, 0.85 mmol) at 0° C. Thereaction mixture was warmed to RT and stirred for 2 h. The volatileswere evaporated under reduced pressure in presence of N₂ atmosphere toafford acid chloride (100 mg, crude). To a stirred solution of acidchloride (100 mg, 0.39 mmol, crude) in DCM (5 mL) were added (6S-FNL-3)(144 mg, 0.47 mmol), N, N-diisopropylethylamine (142 mg, 1.17 mmol) at0° C. The resulting reaction mixture was stirred at RT for 8 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with water (10 mL) and extracted with CH₂Cl₂ (2×20 mL). Combinedorganic extracts were washed by saturated NaHCO3 solution (2×30 mL)followed by citric acid solution (1×20 mL. The separated organicextracts were dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to obtain crude product, which was purified by silica gelcolumn chromatography eluting with 2% MeOH/CH₂Cl₂ to afford compound(6S-FNL-29) (120 mg, 56.8%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ8.00-7.91 (m, 1H), 7.50-7.39 (m, 2H),7.28-7.13 (m, 4H), 5.67 (s, 2H), 5.21-5.16 (m, 1H), 4.44-4.40 (m, 1H),4.20 (d, J=7.6 Hz, 2H), 3.90-3.81 (m, 3H), 3.39-3.34 (m, 1H), 2.50 (s,3H), 2.22-2.15 (m, 2H), 1.92-1.86 (m, 2H), 1.82 (s, 3H), 1.20 (d, J=6.4Hz, 3H);

LCMS m/z: 544.6 [M⁺+1]; HPLC: 98.6% (both enantiomers)

Synthesis of (2R,3S)-4-amino-3-(2-(5-(1-(cyclohexylmethyl)-5-methyl-1H-1, 2,3-triazole-4-carbonyl)-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl)acetamido)-4-oxobutan-2-yl acetate (6S-FNL-30)

To a stirred solution of 6S-BE (200 mg (crude), 0.82 mmol) in DCM (10mL) was added (6S-FNL-3) (270 mg, 0.82 mmol), N, N-diisopropylethylamine(320 mg, 2.48 mmol) at 0° C. The resulting reaction mixture was stirredat RT for 8 h. After consumption of the starting material (by TLC), thereaction mixture was diluted with water (10 mL) and extracted withCH₂Cl₂ (2×30 mL). Combined organic extracts were washed by NaHCO₃solution (1×20 mL), citric acid solution (1×20 mL) followed by brinesolution (2×30 mL). The separated organic layers were dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to obtain crudeproduct, which was purified by silica gel column chromatography elutingwith 2% MeOH/CH₂Cl₂ to afford compound (6S-FNL-30) (86 mg, 19.7%) as anoff-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.98 (dd, J=8.8 Hz, 9.2 Hz, 1H), 7.47 (dd,J=7.6 Hz, 1H), 7.16 (dd, J=7.6 Hz, 10.0 Hz, 1H), 5.22-5.13 (m, 1H),4.43-4.39 (m, 1H), 4.20-4.14 (m, 3H), 4.02-3.99 (m, 1H), 3.87 (t, J=6.0Hz, 4H), 2.48 (s, 3H), 2.20-2.11 (m, 2H), 2.42-2.30 (s, 1H), 1.88-1.85(m, 2H), 1.82 (s, 3H), 1.64-1.59 (m, 4H), 1.48-1.45 (m, 2H), 1.27-1.11(m, 4H), 0.84 (d, J=6.8 Hz, 3H);

LCMS m/z: 532.6 [M⁺+1]; HPLC: 91.2%.

Synthesis of(2R,3S)-4-amino-3-(2-(5-(1-(cyclopentylmethyl)-5-methyl-1H-1,2,3-triazole-4-carbonyl)-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamido)-4-oxobutan-2-ylacetate (6S-FNL-31)

To a stirring solution of 6S-BI (250 mg, 1.19 mmol) in CH₂Cl₂ (10 mL),DMF (0.1 mL) were added oxalyl chloride (0.2 mL, 2.38 mmol) at 0° C. Thereaction mixture was warmed to RT and stirred for 2 h. The volatileswere evaporated under reduced pressure in presence of N₂ atmosphere toafford acid chloride (300 mg, crude). To a stirred solution of acidchloride (300 mg, crude) in DCM (5 mL) was added (6S-FNL-3) (358 mg,1.19 mmol), CVs diisopropylethylamine (0.57 mL, 3.57 mmol) at 0° C. Theresulting reaction mixture was stirred at RT for 1 h. After consumptionof the starting material (by TLC), the reaction mixture was diluted withwater (10 mL) and extracted with CH₂Cl₂ (2×20 mL). Combined organicextracts were washed by brine solution (2×10 mL) and dried overanhydrous Na₂SO₄ concentrated under reduced pressure to obtain crudeproduct, which was purified by silica gel column chromatography elutingwith 2% MeOH/CH₂Cl₂ to afford compound (6S-FNL-31) (100 mg, 16.2%) aspale brown solid.

¹H-NMR: (500 MHz, DMSO-d₆, D₂O): δ5.22-5.19 (m, 1H), 4.39-4.37 (m, 1H),4.22-4.14 (m, 2H), 3.98-3.96 (m, 2H), 3.91-3.85 (m, 2H), 3.38-3.34 (m,1H), 2.50 (s, 3H), 2.41 (s, 2H), 2.36-2.31 (m, 4H), 2.19 (s, 3H), 1.90(d, J=7.5 Hz, 3H), 1.58-1.47 (m, 4H), 1.22-1.16 (m, 4H)

LCMS m/z: 518.6 [M⁺+1];

HPLC: 90% (both enantiomers)

Synthesis ofN-(2-hydroxy-1-(pyrimidin-2-yl)propyl)-2-(1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamide(6S-10)

To a stirring solution of (6S-FNL-20) (1 g, 2.38 mmol) in DCM (20 mL)was added TFA (1.75 mL, 2.38 mmol) at 0° C. and stirred at RT for 2 h.After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure to obtain crude residuewhich was triturated with n-pentane (10 mL) to afford 6S-10 (1 g, crude)as thick syrup was used directly for next step without any purification.

¹H-NMR: (400 MHz, D₂O): 9.10 (d, J=5.2 Hz, 1H), 8.96-8.85 (m, 1H),7.89-7.60 (m, 1H), 4.40-4.22 (m, 1H), 4.12-4.00 (m, 2H), 3.95-3.84 (m,1H), 3.78-3.67 (m, 1H), 3.50-3.44 (m, 2H), 3.20-3.16 (m, 1H), 2.44-2.36(m, 2H), 2.21-2.07 (m, 2H), 1.15-1.12 (m, 3H);

LCMS (ESI): m/z 320.3 [M⁺+1];

Synthesis ofN-(2-hydroxy-1-(pyrimidin-2-yl)propyl)-2-(5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)acetamide(6S-FNL-32)

To a stirring solution of compound 6S-10 (1 g, 2.30 mmol) in DCM (25 mL)was added TEA (0.96 mL, 6.9 mmol) at 0° C. and stirred at RT for 5 min.After added isobutyryl chloride (292 mg, 2.76 mmol) slowly and stirredfor 2 h at RT. After consumption of the starting material (by TLC), thereaction mixture was diluted with water (15 mL). The separated organiclayer was washed with brine solution (1×30 mL). The organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to afford crude compound which was purified by columnchromatography by eluting 10% MeOH/EtOAc to obtained (6S-FNL-32) (230mg, 25.7%) as thick syrup.

¹H-NMR: (500 MHz, CD₃OD): 8.75 (d, J=12.5 Hz, 2H), 7.37 (d, J=10.5 Hz,1H), 4.52-4.45 (m, 1H), 4.22-4.00 (m, 1H), 3.96-3.66 (m, 2H), 3.64-3.57(m, 1H), 3.47-3.33 (m, 3H), 2.84-2.65 (m, 2H), 2.31-2.24 (m, 2H),2.06-1.99 (m, 2H), 1.39-1.24 (m, 3H), 1.19-1.05 (m, 6H);

LCMS (ESI): m/z 390.4 [M⁺+1];

HPLC: 98.4%

Example 12—[³H] MK-801 Binding Assay

Methods

Assays were conducted as described in Moskal et al. (Moskal, J. R., Kuo,A. G., Weiss, C., Wood, P. L., O'Connor Hanson, A., Kelso, S., Harris,R. B., Disterhoft, J. F., 2005. GLYX-13: a monoclonal antibody-derivedpeptide that acts as an N-methyl-D-aspartate receptor modulator.Neuropharmacology. 49, 1077-87) The potentiation of [³H]MK-801 binding(5 nM; 22.5 Ci/mmol) to well washed rat cortical membranes (200 μg) wasmeasured under non-equilibrium conditions (15 min @ 25° C.) in thepresence of increasing concentrations of test compounds and 50 μMglutamate. Zero levels were determined in the absence of any glycineligand and in the presence of 30 μM 5.7 DCKA. Maximal stimulation wasmeasured in the presence of 1 mM glycine, and 50 μM glutamate waspresent in all samples. The facilitation of [³H]MK-801 binding by testscompounds was calculated by using a 3 parameter log agonist vs. responseequation (Graph pad Prism, USA) and potency (EC₅₀ expressed in pM) andmaximal activity (% maximal stimulation) were calculated for the testcompound.

Results

The potency and maximal activity for Compounds B-D and H are as shown inTables 2 and 3 and FIGS. 1-4.

TABLE 2 Activity Compounds pEC50 (pM) (%) B 6 47 C 7 10 D 159 5 H 195 20

TABLE 3 Additional Biological Data Unified Activity Unified UnifiedUnified Data: Activity Activity Activity LTP, Data: Unified UnifiedData: Data: Unified Unified Significant Porsolt Activity ActivityPorsolt [3H] MK-801 MK-801 Activity Data: Activity Data: (S) or FloatingData: Data: Time binding Binding LTP LTP Non- Time Porsolt Porsolt Postassay: EC50 EC50 Augmentation Concentration significant Inhibition DoseDose, Dose Cmpd. (M) (pM) (Percent) (uM) (NS) (Percent) (mg/kg) route(Hours) 6S-FNL-2  1.8E−13 6S-FNL-4 >1e−05 6S-FNL-3 0.00000169 6S-FNL-247.27E−12 110 0.1 S 86 3 IV 1 6S-FNL-7-F2 150 1 S 80 3 IV 1

Example 13—Long Term Potentiation in Hippocampal Slices

Methods

Assays were conducted as described in Zhang et al. (Zhang, X. L.,Sullivan, J. A., Moskal, J. R., Stanton, P. K., 2008. A NMDA receptorglycine site partial agonist, GLYX-13, simultaneously enhances LTP andreduces LTD at Schaffer collateral-CA1 synapses in hippocampus.Neuropharmacology. 55, 1238-50) Sprague-Dawley rats (12-18 days old;Taconic Farms) were deeply anesthetized with isoflurane and decapitated.Rat brains were removed rapidly, submerged in ice-cold artificialcerebrospinal fluid (ACSF, 2-4° C.), which contained (in mM): 124 NaCl,4 KCl, 2 MgSO₄, 2 CaCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, 10 glucose; at pH 7.4,gassed continuously with 95% O₂/5% CO₂). The rat brains were hemisected,the frontal lobes cut off, and individual hemispheres glued usingcyanoacrylate adhesive onto a stage immersed in ice-cold ACSF gassedcontinuously with 95% O₂/5% CO₂ during slicing. Coronal slices (400 μmthick) were cut using a Vibratome (Leica VT1200S), and transferred to aninterface holding chamber for incubation at room temperature for aminimum of one hour before transferring to a Haas-style interfacerecording chamber continuously perfused at 3 ml/min with oxygenated ACSFat 32±0.5° C. Low resistance recording electrodes were made fromthin-walled borosilicate glass (1-2 MΩ after filling with ACSF) andinserted into the apical dendritic region of the Schaffer collateraltermination field in stratum radiatum of field CA1 region to recordfield excitatory postsynaptic potentials (fEPSPs). A bipolar stainlesssteel stimulating electrode (FHC Co.) was placed on Schaffercollateral-commissural fibers in CA3 stratum radiatum, and constantcurrent stimulus intensity adjusted to evoke approximately half-maximalfEPSPs once each 30 s (50-100 pA; 100 μs duration). fEPSP slope wasmeasured before and after induction of LTP by linear interpolation from20 to 80% of maximum negative deflection, and slopes confirmed to bestable to within ±10% for at least 15 min before commencing anexperiment. Bath application of the test compound (1 μM) was applied 30min prior to application of Schaffer collateral stimulus trains toelicit LTP. LTP was induced by stimulation of Schaffer collateral axonswith four high frequency theta burst stimulus trains of 10×100 Hz/5pulse bursts each, applied at an inter-burst interval of 200 ms. Eachtrain was 2 seconds in duration, and trains were applied 15 secondsapart. The signals were recorded using a Multiclamp 700B amplifier anddigitized with a Digidata 1322 (Axon Instruments, USA). Data wereanalyzed using pClamp software (version 9, Axon Instruments) on anIBM-compatible personal computer.

Results

As shown in FIG. 5, Compound B tested at 0.11 μM increased long-termpotentiation after high frequency stimulation of rat Schaffercollateral-evoked NMDA e.p.s.c.s recorded in CA1 pyramidal neurons.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications,websites, and other references cited herein are hereby expresslyincorporated herein in their entireties by reference.

What is claimed is:
 1. A compound represented by formula I:

or a stereoisomer, or an N-oxide, and/or a pharmaceutically acceptablesalt thereof, wherein: R_(b) is H or halogen; R is H or —C(O)CH₃; R₁ isH; R₂ is H; R₃ is C₁-C₆ alkyl; C₁-C₆ alkoxy; —O—C₁-C₆ alkylene-phenyl;or heteroaryl, wherein heteroaryl includes from 5 to 6 ring atoms and 1,2, or 3 of the ring atoms are independently selected from and O; whereinR₃ is optionally substituted with one, two, or three substituentsindependently selected from the group consisting of —NH₂, C₁-C₆ alkyl,hydroxyl, benzyl (optionally substituted with one, two, or threesubstituents independently selected from R^(a)), and —C₁-C₆alkylene-C₃-C₆ cycloalkyl (optionally substituted with one, two or threesubstituents independently selected from halogen and C₁-C₆ alkyl); R^(a)is selected from the group consisting of halogen, C₁-C₆ alkyl and C₁-C₆alkoxy; R₄ is H; R₅ is C₁-C₆ alkyl; X is selected from —C(O)NR^(c)R^(d)and heteroaryl, wherein heteroaryl includes from 5 to 6 ring atoms and1, 2, or 3 of the ring atoms are independently selected from and O; andR^(c) and R^(d) are each or R^(c) and R^(d) together, with the nitrogento which they are attached, form heterocyclyl including from 4 to 6 ringatoms.
 2. The compound of claim 1, wherein R^(b) is H.
 3. The compoundof claim 1, wherein R is —C(O)CH₃.
 4. The compound of claim 1, whereinR₃ is C₁-C₄ alkyl, optionally substituted with one, two or threesubstituents independently selected from —NH₂ and hydroxyl.
 5. Thecompound of claim 1, wherein R₃ is —OC(CH₃)₃.
 6. The compound of claim1, wherein R₃ is —O—CH₂-Ph.
 7. The compound of claim 1, wherein R₃ isheteroaryl including 5 ring atoms, wherein 2 or 3 of the ring atoms areN, and each ring nitrogen is optionally substituted with a substituentindependently selected from methyl, benzyl (optionally substituted withone or two substituents independently selected from F, —OCH₃, and —CH₃),and —CH₂C₆H₁₁.
 8. The compound of claim 1, wherein R₄ is H.
 9. Thecompound of claim 1, wherein R₅ is —CH₃.
 10. The compound of claim 1,wherein X is —C(O)NR^(c)R^(d), wherein each of R^(c) and R^(d) is H. 11.The compound of claim 1, wherein X is —C(O)NR^(c)R^(d), wherein R^(c)and R^(d) together, with the nitrogen to which they are attached, formheterocycyl including from 5 to 6 ring atoms.
 12. The compound of claim1, wherein X is heteroaryl, wherein hereroaryl includes from 5 to 6 ringatoms, and 1, 2 or 3 of the ring atoms are independently selected from Nand O.
 13. A method of treating neuropathic pain, the method comprising:administering to a patient in need thereof a compound represented byformula (I):

or a stereoisomer, or an N-oxide, and/or a pharmaceutically acceptablesalt thereof, wherein: R_(b) is selected from the group consisting of H,halogen, hydroxyl, cyano and C₁-C₆alkyl; R is H, C₁-C₆ alkyl, or—C(O)—C₁-C₆alkyl; R₁ is H or C₁-C₆ alkyl; R₂ is H or C₁-C₆ alkyl; R₃ isC₁-C₆ alkyl; C₁-C₆ alkoxy; —O—C₁-C₆ alkylene-phenyl; C₂-C₆ alkenyl;C₂-C₆ alkynyl; C₃-C₆ cycloalkyl; phenyl; or heteroaryl, whereinheteroaryl includes from 5 to 6 ring atoms and 1, 2, or 3 of the ringatoms are independently selected from the group consisting of N, O, andS; wherein R₃ is optionally substituted with one, two, or threesubstituents independently selected from the group consisting of —NH₂,halogen, C₁-C₆ alkyl, C₁-C₆ alkoxy, hydroxyl, phenyl (optionallysubstituted with one, two, or three substituents independently selectedfrom R^(a)), benzyl (optionally substituted with one, two, or threesubstituents independently selected from R^(a)), and —C₁-C₆alkylene-C₃-C₆ cycloalkyl (optionally substituted with one, two or threesubstituents independent selected from halogen and C₁-C₆ alkyl); R^(a)is selected from the group consisting of halogen, C₁-C₆ alkyl(optionally substituted with one, two or three halogens), C₃-C₆cycloalkyl (optionally substituted with one, two or three halogens), andC₁-C₆ alkoxy (optionally substituted with one, two or three halogens);R₄ is H or C₁-C₆ alkyl; R₅ is H or C₁-C₆ alkyl; X is selected from thegroup consisting of H; C₁-C₆ alkyl; hydroxyl; C₁-C₆ alkoxy; —CO₂H;—C(O)NR^(c)R^(d); and heteroaryl, wherein heteroaryl includes from 5 to6 ring atoms; 1, 2 or 3 of the ring atoms are independently selectedfrom the group consisting of N, O, and S; and heteroaryl may beoptionally substituted with one, two, or three substituentsindependently selected from the group consisting of halogen, C₁-C₆alkyl, C₁-C₆ alkoxy, hydroxyl, and phenyl; and R^(c) and R^(d) are eachindependently selected from the group consisting of H, C₁-C₆ alkyl andphenyl; or R^(c) and R^(d) together, with the nitrogen to which they areattached, form heterocyclyl, wherein heterocyclyl includes from 4 to 6ring atoms but not more than two ring heteroatoms (including thenitrogen atom attached to R^(c) and R^(d)), and the second ringheteroatom, when present, is independently selected from the groupconsisting of N, O, and S; and heterocyclyl is optionally substitutedwith one, two or three substituents independently selected from thegroup consisting of halogen, cyano, oxo, and C₁-C₆ alkyl.
 14. The methodof claim 13, wherein R_(b) is H; R is H or —C(O)CH₃; R₁ is H; R₂ is H;R₄ is H; and R₅ is CH₃.
 15. The method of claim 13, wherein R₃ is C₁-C₄alkyl; C₁-C₄ alkoxy; —OCH₂-phenyl, or heteroaryl, wherein heteroarylincludes from 5 to 6 ring atoms and 1, 2 or 3 of the ring atoms are N;and wherein R₃ is optionally substituted with one, two or threesubstituents independently selected from —NH₂, C₁-C₄ alkyl, hydroxyl,benzyl (optionally substituted with one, two or three substituentsindependently selected from halogen, C₁-C₄ alkyl, and C₁-C₄ alkoxy), and—CH₂—C₃-C₆ cycloalkyl.
 16. The method of claim 13, wherein X is selectedfrom —C(O)NR^(c)R^(d) and heteroaryl, wherein heteroaryl includes from 5to 6 ring atoms and 1, 2 or 3 of the ring atoms are N or O; and each ofR^(c) and R^(d) is H, or R^(c) and R^(d) together, with the nitrogen towhich they are attached, form heterocycyl including from 5 to 6 ringatoms.
 17. The method of claim 13, wherein the neuropathic pain isacute.
 18. The method of claim 13, wherein the neuropathic pain ischronic.
 19. The method of claim 13, wherein the neuropathic pain isassociated with fibromyalgia.
 20. The method of claim 13, wherein theneuropathic pain is associated with diabetic neuropathy.